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Restriction Enzymes as a Target for DNA-Based Sensing and Structural RearrangementClick to copy article linkArticle link copied!
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Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States
*E-mail: [email protected]
Abstract
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DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution.
Copyright © 2018 American Chemical Society
Introduction
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Sensors in their most basic form are devices that recognize a target and transduce that detection event into a signal that can be read and interpreted by an end user. A variety of sensors exist to accommodate a range of target types with chemical and biological targets being of increased interest. One challenge in particular for biosensing is interacting with the target using a probe that is relatively of the same size. Nanoscale structures have this advantage given many biomolecules are on the order of a few nanometers. (1-10) DNA nanostructures, pioneered by Seeman, (11) use the binding of Watson–Crick base pairs to program specific, non-native formations that can then perform various tasks at the nanoscale, and this technology is particularly suited for biosensing because it is a native biological material. (12-15) DNA nanostructures have been shown to be highly modular for the creation of a range of two-dimensional and three-dimensional motifs, including both static and dynamic structures. (16-19) The work within dynamic systems has often focused on induced motion through competitive DNA hybridization, such as multistep logic, (20) hybridization chain reaction, (21) DNA tweezers, (22) and hinged lid boxes. (23) This is a highly adaptable design regime that allows for rapid switching of states and a nearly limitless range of possible sequences to use. The strategy however is less adapted for non-nucleic acid sensor targets. The ability to adapt the modularity of DNA structural design to non-DNA molecules, such as proteins and enzymes, represents an area of interest and recent development.
Motifs, such as aptamers, can be easily added to other DNA structures, but their shape response is limited to a specific reaction phenotype. (24-26) In a similar manner, DNAzymes use DNA/RNA chimeric sequences and act as a catalytic reaction center, allowing for the dynamic adjustment of the structure for both structural rearrangement. (27-31) There has been minimal work, however, interacting DNA structures directly with enzymatic inputs for either sensing or directed structural rearrangement. Zuo et al. demonstrated a molecular beacon approach that uses exonuclease to generate an amplified fluorescent output, but this is neither the sensing target nor a means to structural change. (32) It is our intent to explore the use of restriction enzymes as a directing molecule for the development of DNA-based sensors and enzyme-directed machines. We hypothesize that integration with these enzymes will enable multistep sensing.
Restriction enzymes, also called restriction endonucleases, are enzymes that cut DNA at specific sequences. Naturally found in bacteria to defend against viral pathogens, restriction enzymes have been harnessed by researchers and have proven a powerful asset for use in biotechnology applications, such as DNA cloning. These enzymes typically recognize sequences of DNA between 4 and 8 base pairs and can cut double-stranded DNA in a staggered manner, leaving a single-stranded overhang (sticky end) or they can cut at the same place on each strand producing a blunt end. (33-35)
Our previous efforts with DNA sensors produced a multiarm switch that enabled a logic-capable photonic output for three simultaneous single-stranded DNA targets. (36) Linkers joining the arms can be added or removed by the addition of specific DNA sequences. This switch uses a base set of arms, each of which contains the molecular dye. From this base, the switch can be assembled with a range of different dyes and different linker lengths to produce a vast range of optical output. (37) This modularity makes it an ideal candidate to explore restriction enzyme-enabled sensing and rearrangement. Although we have explored many dye triads as well as structural modification, to date, only DNA inputs have been used to modify the optical response. In this work, we investigate the role of restriction endonuclease to modulate our three-dye optical network. We test six different restriction enzymes with both sticky and blunt-end cleavage types and combine three of these into a single device capable of detecting rearrangement via single, double, and triple enzymatic digestion. The cleavage also results in the release of a seven base toehold, which we demonstrate can be used for the one-pot rearrangement and inclusion of a Cy5-containing DNA.
Results
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Structural Design
The three-arm switch, detailed in previous publications, (36, 37) was used as a basis for the underlying design of the enzyme-responsive DNA structure. This DNA-based structure positions three covalently linked fluorescent dyes and utilizes their overlapping excitation and emission profiles to form a Förster resonance energy transfer (FRET) triad that is responsive to changes in distance between each of the three fluorophores. This structure provides the demonstrated ability to detect three separate targets within one device, thus enabling complex detection. (36) The underlying DNA structure consists of three double-crossover junctions formed by an arm strand annealed with a capping strand with a molecular dye covalently attached at the central end position of the arm strand. Each of these crossovers represents one of three arms, Arm1, Arm2, or Arm3, and are linked together via DNA linkers, which position the attached dyes into the FRET triad.
The controlling element in the device is the presence of an intact linker strand, which brings the dyes into proximity and turns on the FRET interaction. In Buckhout-White et al., (36) the linkers used were single-stranded and could be removed through toehold-mediated strand displacement, thus rendering this device a DNA sensor. With our goal to move into more complex, non-DNA targets, we have modified the original design of the linkers to include a double-stranded recognition sequence for restriction enzymes. This modification uses the basic structure detailed above, including the three-arm crossover structures, but replaces the single-stranded linker with a 10 base pair double-stranded linker, a long linker strand with a short 10 base compliment strand, which serves as the cleavage site. The overall structure is depicted in Figure 1A.
Figure 1
Figure 1. (A) Schematic of the DNA switch structure and mechanism for rearrangement via enzymatic cleavage: (i) partial structure schematic indicating the arms and cleavage site. Each of these structures contains two molecular dyes, and the cleavage is transduced by simple FRET behavior; (ii) full three-arm structure, which contains three dyes and is transduced through examining each of the three acceptor-to-donor ratios. The linker between each arm of the structure is a 10 base double-stranded DNA and is coded to be a cleavage site for a specified restriction enzyme. The cleavage of one or more of these linker regions allows for the separation of the fluorescent dyes, which reduced the output of the donor-to-acceptor ratio. (B) Sequence of one of three linker sequences with the restriction site in bold and the cutting location indicated by the triangles. (C) Emission and excitation profiles of the Cy3, Cy3.5, and Cy5 molecular dyes. (D) Spectral overlap of these dyes form the basis of the multi-FRET-based optical output.
The modularity of the switch design allows for the structure to be assembled partially such that only two dye-containing arms are present, connected by one linker containing the recognition area, thus greatly simplifying the structure to allow for iterative analysis of the individual components. The partial structure used for testing each enzyme independently is depicted in Figure 1A,i, which shows one of the three dye pairs that can be formed using the partial structure: Cy3–Cy5, Cy3–Cy3.5, and Cy3.5–Cy5. The rearrangement induced by the cleavage from the restriction enzyme is shown on the full three-arm structure in Figure 1A,ii. The same process would proceed on the partial structure except the two arms would fully dissociate as no remaining DNA is left to bind the structure.
Each linker region is coded with a recognition site for a unique restriction enzyme. Six enzymes in total were tested: XbaI, EcoRI, BamHI, NcoI, SmaI, and XhoI. Figure 1B shows the sequences of all six restriction enzymes that were tested. Of these enzymes, XhoI, EcoRI, and SmaI were found to work in concert within a fully assembled switch. The bold portion of the sequence represents the restriction site, and the arrows indicate the cutting location. As can be seen in Figure 1B, we investigated both blunt-end cleavage and sticky-end cleavage. The sequences and melting temperatures for all oligos are listed in Table S1 in the Supporting Information (SI).
The transduction functionality of this switch is based on the cyclic FRET behavior of three spectrally overlapping and closely spaced molecular dyes. This is discussed in detail in Buckhout-White et al. (36) The dye triad used for these studies were Cy3, Cy3.5, and Cy5. Figure 1C shows the excitation and emission curve for each dye, and the spectral overlap is shown in Figure 1D. The Förster distance, the distance which corresponds to a theoretical 50% transfer efficiency, is 5.3 nm for Cy3–Cy3.5, 5.4 nm for Cy3–Cy5, and 6.0 nm for Cy3.5–Cy5. For the original work, a spacing of nine bases, or ∼3 nm, would have theoretically yielded ∼95% efficiency. In practice, we measured 30% efficiency, which was sufficient to demonstrate change in the full three-arm configuration. This 10 base linker will have a 3.5 nm spacing, but will reduce some of the torsional contribution given the spacing is on par with the length of one full helical turn of DNA. As such, we expect similar performance from this configuration.
Although the activity of restriction enzymes is well documented and their use considered routine for many genetic engineering protocols, our application explores the limits of double-stranded DNA size and temperature conditions, for which these enzymes are optimized. To retain the close proximity of the dye molecules in the uncut “off” state, it is necessary to minimize the total length of the double-stranded linker. The recognition sites of many restriction enzymes, including those we worked with, are six bases, and linkers were designed to include two additional bases on either side of the recognition site. According to the enzyme manufacturers, it is recommended to include a minimum of six (38) additional bases flanking the restriction site for optimal enzyme performance. The 10 base double-stranded linker is formed using a 10 base compliment to the single-stranded linker strand and thus naturally has a melting temperature ranging from 41.5 to 57.7 °C. The melt temperature defines the point at which half of the DNA is single-stranded. With 41.5 °C being the low range, we prefer that the enzyme be able to work at room temperature. Although several restriction enzymes, including SmaI, cut their recognition site at room temperature (25 °C), most restriction enzymes perform optimally at 37 °C. Due to the requirement that the enzymes function at room temperature and cut a 10 base pair DNA linker, it was critical to be able to test each restriction enzyme independently to determine which would function under these nonideal conditions.
Single-Enzyme Cleavage
Partial structures containing two arms and a single recognition sequence were used to assess the ability of each enzyme to cleave the 10 base pair linker at room temperature. The activity of each of the six enzymes, XhoI, NcoI, SmaI, XbaI, BamHI, and EcoRI, was compared against a positive and a negative control. The negative control comprised the partial structure without any enzyme present. This represents the condition with no enzymatic cleavage of the linker. The positive control is an approximation of a full cleavage by the enzyme and is represented experimentally by forming structures that lack the linker connecting the two dye-containing arms. The restriction enzymes XbaI, XhoI, NcoI, and SmaI were tested using the Arm1–Cy3, Arm2–Cy3.5 partial structure. EcoRI was tested using the Arm2–Cy3.5, Arm3–Cy5 partial structure, and SmaI and BamHI were tested using the Arm1–Cy3, Arm3–Cy5 partial structure. For all enzymes that were considered, five enzymes in four unique combinations emerged, showing ideal cutting behavior and no nonspecific cutting behavior in the presence of a nontargeted enzyme. Of the four combinations, NcoI-BamHI-XhoI, NcoI-EcoRI-XhoI, XhoI-SmaI-BamHI, and XhoI, EcoRI-SmaI, the latter was chosen to perform the full structured variations.
Figure 2A shows the analyzed fluorescent data, whereas Figure 2B shows the gel electropherogram. For each of the characterization methods, the samples were prepared in the same way in parallel runs with the positive and negative controls. The positive control solution was annealed according to the protocol specified in the Methods section in a batch large enough for 24, 20 μL sample to be produced. This sample contained equal molar ratios of each of two dye-containing arm strands and their corresponding capping strands as well as the linker and the compliment to the linker, which provides for the double-stranded cleavage site all in the provided CutSmart buffer. With CutSmart being a nonstandard buffer for DNA formation, a formation analysis was performed prior to begin the enzyme cutting and can be seen in the Supporting Information (SI) (Figure S1). Each of these samples was aliquoted into a 384-well plate in triplicate, where 2 μL of the specified enzyme was added and then mixed by gentle pipetting. For the negative control, the solution was annealed separately without the linker strand or its compliment. Both the positive control and negative control added 2 μL of buffer to account for the volume of the absent enzyme. The samples were allowed to digest for 1 h minimum before measuring the fluorescent output. All fluorescent measurements were excited at a wavelength of 515 nm, consistent with the shoulder of the Cy3 excitation peak and recorded between 530 and 800 nm. These parameters were set as such to allow for consistent readout once all three dyes were used in the full system. The samples for the gel electrophoresis were taken from the plate after the fluorescent data were obtained and mixed with the gel-loading buffer before pipetting the samples into the 3% agarose gel.
Figure 2
Figure 2. Data from single-enzyme partial structures. (A) Ratios of the acceptor dye over the donor dye photoluminescence (PL) peak values. The three plots shown each represent a different programmed sequence designed for the enzyme specified. Each sequence was tested against six different enzymes with a negative, no enzyme, and positive, no linker, control. (B) Gel electropherogram on the partial structure before and after introduction of the specific enzyme.
To assess the efficacy of the cleavage via fluorescence, the maximum value of the deconstructed area of the acceptor peak in each of the dye pair is divided by the deconstructed donor peak maximum. XhoI and SmaI were initially analyzed using the Arm1–Cy3, Arm2–Cy3.5 partial structure and thus the ratio shown for these constructs is Cy3.5/Cy3. For EcoRI, the Arm2–Cy3.5, Arm3–Cy5 partial structure was used and the ratio is thereby Cy5/Cy3.5. These fluorescent PL ratios are plotted in Figure 2A for the three enzymes chosen for the triad assembly. The data for the enzymes not selected and the table showing all possible dye triads can be seen in the SI (Figure S2 and Table S2). The gray bars, first and last, in Figure 2A, represent the controls for each system, with the negative control indicating the maximum value for no enzyme activity and the positive control approximating a complete cleavage of the linker. All enzymes are shown, and based on fluorescent data alone, it is clear that these three enzymes show cutting behavior when presented with the specified enzyme and show no cutting behavior when presented with the nonspecified enzyme. It is of note that the value of the specified enzyme does not quite reach the value of the negative control. This may be due to a very small amount of incomplete digestion or may just reflect the fact that the negative control is an approximation because no portion of the linker or compliment is present.
Figure 2B shows the gel migration of the control sample as well as the specified enzyme digestion for the partial switch structure. The primary band at about 240 bp according to the ladder represents the fully formed structure. Once the enzyme is added, this band increases its migration consistent with 150 bp, roughly half. The intensity of the band also increases due to the doubling of the mass of DNA at that specific size. There is a small remnant of the full-structure band that is consistent with the thought that we are getting close to but not 100% digestion of these structures. This may also be a result of potential rehybridization of the sticky ends, or reassociation of the blunt ends that occur after the cleavage. In all, both characterization methodologies support the same conclusion that these three enzymes exhibit good cleavage with little to no nonspecific activity.
Kinetics of Enzyme Activity
With the activity confirmed, we assessed the kinetic rate of the enzymatic cleavage. To reiterate, these enzymes are demonstrating cleavage under nonideal conditions. The cleavage for all experiments occurs at room temperature, with most of these enzymes optimized for 37 °C. The cleavage site is also contained on a 10 base oligo with three-way junctions at both ends of that oligo. For these enzymes, it is recommended that there be a minimum of six (38) base pairs on either side of the cleave site. Figure 3 shows the kinetic curves produced by measuring the same acceptor-to-donor ratio for each of the partial structures. This measurement was recorded every 20 min, with the exception of the first time point, which occurred 10 min after the initial plating. The black bars flanking the curves are averaged values of controls, positive, and negative, taken over the entire time. The variations in position of the positive and negative controls are directly due to the photophysical properties of the dyes in each system. The sharp line in the EcoRI indicated that full cleavage occurs within the 10 min time frame between initial plating and the addition and mixing of the enzyme.
Figure 3
Figure 3. Assay of kinetic rate of cleavage for the partial structure cleavage. The top line in each plot represents the average of the positive control, and the bottom line is the average of the negative control.
Three-Arm Switch Performance
With the three enzymes determined to operate efficiently in the partial structure, we placed the three chosen enzymes’ recognition sequences into each of the three linker regions; XhoI in linker 1 between Cy3 and Cy3.5, EcoRI in linker 2 between Cy3.5 and Cy5, and SmaI in linker 3 between Cy3 and Cy5. Using the cutting of one linker as an input, we find eight potential permutations for the full three-arm switch. This is equivalent to the same eight potential permutations in Buckhout-White et al. (36) The difference with the enzyme switch is the fragments of the cleaved linker is still present whereas the linker removal via strand displacement from the previous work, removes the entire sequence. The corollary between these two systems allows us to provide a set of positive controls such that we are mimicking the cleavage of one, two, or three of the linkers by removing the linker altogether.
To test the three-arm switch, we created a series of single, double, and triple digestions and compared them with the corresponding linker-removed structures. For example, the corollary to XhoI presence would be the removal of linker 1. For this series, we used XhoI single digestion, EcoRI single digestion, XhoI and EcoRI double digestion, and then the final XhoI EcoRI and SmaI triple digestion. For the three-arm structure, the FRET pathways are all interrelated and it is necessary to examine all three acceptor-to-donor ratios to get a full understanding of the spectral output. Figure 4 displays the acceptor-to-donor ratios for the enzyme digestion (A) and the linker-removed corollaries (B). Comparison shows general agreement between the two sets. As expected from the partial structure digestions, we do not fully reach the level of the positive controls. This again may be due to partial cutting, reassembly of the cut end, or inherent anomalies between a fully cut structure and the linker-removed version. It may also be due to the amount of glycerol in the system from the double and triple digestions. We chose to keep all variables constant, which meant the amount of glycerol inserted into the system was 10% for the double digestion and 15% for the triple digestion. Although this does not eliminate the desired reaction, it may limit completion of the reaction or full rearrangement of the structure. If we look at the trends of each ratio between samples, we see that in the case of the Cy3.5/Cy3 ratio the value decreases slightly, increases, and then subsequently decreases in the next two samples. All ratios follow the same trends with the exception of the Cy5/Cy3.5 ratio between the negative control and the XhoI enzyme addition. Here, the value roughly stays level in the enzyme sample and increases in the control. These similar trends can also be seen if we plot the dye contribution as a percentage of the total area. The ternary plot shown in Figure S3 displays this presentation.
Figure 4
Figure 4. Plots of the three acceptor-to-donor ratios for each of the full three-arm structures. (A) Single-, double-, and triple-enzyme digest for the three-arm structures. (B) Comparable control structures in which a linker is removed where the corresponding enzyme structure would be cleaved.
Structural Rearrangement
One added benefit of the 10 base length of the recognition site compliment is that upon the sticky-end cleavage realized by both the XhoI and EcoRI enzymes we are left with a 7 base, 3 base split of the 10 base sequence. The three bases that remain of the linker strands do not have a sufficient melt temperature to allow this duplex to remain stable and thus the seven bases of the compliment dissociate into solution. The same is true of the other side where the three bases of the compliment do not have the energy to remain associated with the seven base linker strand. This end thereby leaves a seven base strand that is now suitable as a toehold for additional structural modification. This process is depicted schematically in Figure 5A. It is important to note that this toehold only exists after the restriction enzyme has fully cleaved the target sequence.
Figure 5
Figure 5. (A) Schematic illustration of the partial structure enzyme cleavage and rearrangement via addition of an additional Cy5-containing DNA oligo. (B) PL Intensity of each of the structures depicted in (A), with XhoI + oligo and XhoI + bridge both being one-pot reactions. (C) PL peak height plots for each of the three dyes in the system. The addition was performed both as a one-pot reaction and a sequential reaction. The controls of the switch plus the oligo or bridge are done in the absence of enzyme and show little to no reaction without the presence of the enzyme.
To utilize this toehold display, we have formed the three-arm structure in full and partial form, with only the Cy3 and Cy3.5 dyes present on Arm1 and Arm2, respectively. For the full structure, Arm3 is present but in an unlabeled form to preserve the structural integrity but allow for the use of the Cy5 dye for our new addition. For this reconfiguration, we have designed two versions of a Cy5-labeled DNA that will interact with the exposed toehold. The oligo version is a simple single-stranded DNA of 15 bases with a Cy5 that will have the closest interaction with the Cy3. The bridge version is a partially duplex DNA with two identical binding regions that will bridge two of the three arm switches together. This is depicted in the bottom panel of Figure 5A.
Figure 5B shows the spectra of the original partial two-arm structure, the structure with the XhoI enzyme added, XhoI plus the bridge DNA, and XhoI plus the oligo DNA. In comparing the XhoI spectra to that where either the bridge or the oligo is added, there is a clear decrease in the Cy3 peak at 550 nm and a clear increase in the Cy5 peak at 650 nm. This change alone shows the inclusion of the new oligo, thus demonstrating the ability to modify the structure after the enzymatic cleavage has taken place.
Figure 5C shows further analysis in which each of the three acceptor-to-donor ratios is plotted. The control is shown in section I and is derived from the data from “original” in Figure 5B. Section II shows two controls where the oligo and the bridge are added to the control without enzyme. For the bridge structure, we see no increase in Cy5, but for the oligo structure, we see a slight increase in the Cy5 excitation, indicating some leakage. Section III is where XhoI is added, and we see the clear increase in the Cy3 excitation. For sections IV and V, we distinguished between a sequential addition method, where the enzyme is added to the full structure and allowed to cleave and then either the bridge or the oligo is added, and the one-pot addition, where the oligo or bridge and the enzyme are added simultaneously. In each case, the oligo or bridge is added in a 4-fold molar excess to the switch. For the sequential addition, we see minor increase from the oligo Cy5/Cy3 excitation signal but similar values with the other two ratios. For the one-pot addition, the bridge addition shows slight decrease in both the Cy5/Cy3 signal and the Cy3.5/Cy3 signal. Although the lower Cy5/Cy3 signal indicates a potentially lower rate of binding, the lower Cy3.5/Cy3 ratio may mean more disruption of the Cy3–Cy3.5 transfer. In all, this may be a direct result of the bridge molecule having asymmetric dye position of the Cy5.
Discussion and Conclusions
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In this work, we utilized the inherent fit with restriction endonuclease to expand the range of detectable materials outside of the realm of simple DNA. We have clearly demonstrated the ability of a three-arm switch to be applied to enzymatic inputs. Furthermore, we have demonstrated that these enzymes can be used to rearrange the structure of the DNA, allowing multistep processes to occur, a necessary step toward the evolution of complex sensing. In investigating the use of restriction enzymes in this confined parametric space, we have also demonstrated the ability of these materials to engage effectively outside their optimal environment. We see no difference in cleavage rate between that of blunt-end- and sticky-end-type cleavage sites. In the steady-state measurement, we see no distinction in the cleavage efficiency between these cleavage types either. However, in the kinetic assay, the SmaI enzyme, a blunt-end restriction enzyme, completes the reaction with a higher ratio, indicating less complete cleavage than the control compared to either of the other two enzymes, XhoI or EcoRI, both of which are sticky-end cleavage types. This difference may relate to the affinity for the blunt-end nonspecific adhesion, which is documented in ref 39 or may be an artifact of the measurement parameter. Further work on a broader array of both cleavage type enzymes will help alleviate this question and expand the library of enzymes applicable to this type of activity environment.
We have presented the transfer from the partial structure analysis to the full-structure analysis and the multiplexing ability that this brings, as demonstrated by the successful single, double, and triple digestion. Although these do not reach their full-potential on–off range as demonstrated by the linker-removed controls, we still see clear distinction enough to determine which enzymes are acting. As demonstrated by the unique change particularly seen in the addition of the EcoRI enzyme, it is possible to determine, blindly, which enzyme is acting. Improvement on this may be available through dye change or condition optimization. Further, as a sensing modality, this multiplexing ability is quite powerful and may not be limited to simply three enzymes. The modular structure could easily be made more complex by the addition of arms to the unit. We have also shown that it can operate distinctly using different dye triads and that there are several unique combinations of enzymes. (37) This means that we may potentially be able to have two separate switches in solution, each with unique recognition sites and optical output.
The structural rearrangement offers perhaps the most unexpected outcome of this work. As a byproduct of restriction enzyme cleavage of the target DNA, we reveal an active DNA toehold. Multistep reactions are not new within the field of DNA nanotechnology. Concentric FRET systems based on protease cleavage and quantum dot assembly show similar optical performance with regard to multienzyme detection, but they are limited to simple detection mechanisms. (40) Other work demonstrates the ability to interact with an existing structure and expose a toehold that will continue the reaction in a prescribed manner. (41-43) Our present demonstration, however, shows the clear ability of enzyme-directed chain reactions within a confined structural environment. This is evidenced by the appearance of the Cy5 excitation peak, which is not seen in either the control or the original structure. The effect seen in both the sequential and one-pot demonstrations further demonstrates that the secondary rearrangement with the toehold is only activated once the enzymatic cleavage has occurred.
With such little leakage seen, this simple demonstration portends to much greater potential application. The harnessing of multienzyme, multistep assemblies could have an impact on the fields of DNA nanorobotics and enzyme-directed nanofabrication. Within the context of complex sensing and theranostics, it is also conceivable that these systems may be adapted to a larger cagelike structure that upon interaction with the target enzymes can release payload or rearrangement to deliver targeted DNA codons.
In all, we have expanded the utility of the logic FRET-enabled three-arm DNA switch to the detection of restriction enzymes. We have shown six different enzymes in all and three enzymes in detail. These three enzymes have clear cleavage efficiency and no nonspecific cleavage, a necessary requirement for this three-target system to function. We have demonstrated single, double, and triple digestions with clear photonic distinction in the output, a necessary criterion to determine which enzymes are present. Finally, we show the ability of these enzymes to be used as triggers for site-specific rearrangement of these DNA structures, which may have broad-reaching potential for complex sensing and smart nanoscale systems.
Methods
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DNA
The DNA sequences are based on Buckhout-White et al. (36) with the exception of the linker sequences, which are designed to include cleavage sites for the specified restriction enzymes. All unlabeled and Cy3- and Cy5-labeled DNA are synthetic oligos ordered from Integrated DNA Technologies (Coralville, IA). The Cy3.5-containing oligos are sourced from Eurofins genomics (Louisville, KY). The oligos used were diluted to 20 μM working concentrations in water and analyzed for concentration using Thermo Fisher NanoDrop 2000.
Structural Assembly
All of the partial and full structures were assembled in a total volume of 80 μL with a 0.5 μM concentration unless specified otherwise. A small volume of 2 μL of each 20 μM DNA oligo was added to form either the partial or full structures. The CutSmart (New England Biolabs) buffer (8 μL) was used in each 80 μL sample unless specified otherwise. Each 80 μL sample was annealed on a ProFlex Thermal cycler using a program that heats the sample up to 95 °C for 5 min and is ramped down 1 °C every minute until 4 °C, at which temperature it is held.
Enzyme Digestion
All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.
Structural Rearrangement
For the structural rearrangement studies, Cy5 oligo and Cy5 bridge were prepared in a 20 μM dilution in the CutSmart buffer and annealed using the same thermal ramp protocol used to assemble the switch structure. A small volume of 1 μL of this preannealed solution was used for each addition, which represents a 4-fold molar excess to the assembled structure. The oligo is allowed to react with the switch structure at room temperature for a minimum of 10 min.
FRET Data Collection and Analysis
Fluorescence data were collected using the Tecan Infinite M100 dual monochromator multifunction plate reader that has a xenon flash lamp (Tecan, Research Triangle Park, NC). A small volume of 20 μL of each 0.5 μM concentration sample was inserted into a single well in a 384-well plate. All of the samples were run in triplicate unless otherwise specified. The DNA dye-labeled samples were excited at 515 nm. The data were recorded over an emission ranging from 530 to 800 nm. The raw data were deconstructed using model Cy3, Cy3.5, and Cy5 emission spectra. The maximum peak value of each spectra corresponding to 556, 606, and 664 nm was used to produce the donor-to-acceptor ratio values.
Gel Electrophoresis Data Collection and Analysis
Three percent agarose gels made with 1× tris acetate EDTA buffer were used to collect the gel electrophoresis data unless otherwise indicated. For every 100 mL of gel solution, 20 μL of GelRed stain (Biotium; Fremont, CA) was used. A DNA ladder (BioMarker EXT Plus, Bioventures Inc; Murfreesboro, TN) was used to determine the length of the double-stranded DNA bands. A volume of 10 μL of a 12 μL sample that consisted of 10 μL of digested sample (or control) and 2 μL of a loading dye was loaded into each well; 6 μL of ladder was inserted into one well that consisted of 5 μL of ladder and 1 μL of loading dye unless otherwise indicated. Each gel experiment was run at 80 V for 1 h. The Bio-Rad ChemiDoc XRS+ System was used to analyze the gel electrophoresis data.
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01333.
DNA sequences, comparative buffer analysis, full enzyme analysis, enzyme triad table, and ternary plot for three dye assemblies (PDF)
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Author Information
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- Susan Buckhout-White - Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States;
http://orcid.org/0000-0003-0524-1543;
- Igor L. Medintz - Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States;http://orcid.org/0000-0002-8902-4687
Acknowledgment
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Financial support from NRL and NRL-NSI is gratefully acknowledged.
References
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This article references 43 other publications.
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- 2Haes, A. J.; Van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 10596 DOI: 10.1021/ja020393xGoogle Scholar2A nanoscale optical biosensor: Sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticlesHaes, Amanda J.; Van Duyne, Richard P.Journal of the American Chemical Society (2002), 124 (35), 10596-10604CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Triangular silver nanoparticles (∼100 nm wide and 50 nm high) have remarkable optical properties. In particular, the peak extinction wavelength, λmax of their localized surface plasmon resonance (LSPR) spectrum is unexpectedly sensitive to nanoparticle size, shape, and local (∼10-30 nm) external dielec. environment. This sensitivity of the LSPR λmax to the nanoenvironment has allowed us to develop a new class of nanoscale affinity biosensors. The essential characteristics and operational principles of these LSPR nanobiosensors will be illustrated using the well-studied biotin-streptavidin system. Exposure of biotin-functionalized Ag nanotriangles to 100 nM streptavidin (SA) caused a 27.0 nm red-shift in the LSPR λmax. The LSPR λmax shift, ΔR/ΔRmax, vs. [SA] response curve was measured over the concn. range 10-15 M < [SA] < 10-6 M. Comparison of the data with the theor. normalized response expected for 1:1 binding of a ligand to a multivalent receptor with different sites but invariant affinities yielded approx. values for the satn. response, ΔRmax = 26.5 nm, and the surface-confined thermodn. binding const. Ka,surf = 1011 M-1. At present, the limit of detection (LOD) for the LSPR nanobiosensor is found to be in the low-picomolar to high-femtomolar region. A strategy to amplify the response of the LSPR nanobiosensor using biotinylated Au colloids and thereby further improve the LOD is demonstrated. Several control expts. were performed to define the LSPR nanobiosensor's response to nonspecific binding as well as to demonstrate its response to the specific binding of another protein. These include the following: (1) electrostatic binding of SA to a nonbiotinylated surface, (2) nonspecific interactions of prebiotinylated SA to a biotinylated surface, (3) nonspecific interactions of bovine serum albumin to a biotinylated surface, and (4) specific binding of anti-biotin to a biotinylated surface. The LSPR nanobiosensor provides a pathway to ultrasensitive biodetection expts. with extremely simple, small, light, robust, low-cost instrumentation that will greatly facilitate field-portable environmental or point-of-service medical diagnostic applications.
- 3Liu, S. P.; Su, W. Q.; Li, Z. L.; Ding, X. T. Biosens. Bioelectron. 2015, 71, 57 DOI: 10.1016/j.bios.2015.04.006Google Scholar3Electrochemical detection of lung cancer specific microRNAs using 3D DNA origami nanostructuresLiu, Shuopeng; Su, Wenqiong; Li, Zonglin; Ding, XiantingBiosensors & Bioelectronics (2015), 71 (), 57-61CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)Recent reports have indicated that aberrant expression of microRNAs is highly correlated with occurrence of lung cancer. Therefore, highly sensitive detection of lung cancer specific microRNAs provides an attractive approach in lung cancer early diagnostics. Herein, we designed 3D DNA origami structure that enables electrochem. detection of lung cancer related microRNAs. The 3D DNA origami structure is constituted of a ferrocene-tagged DNA of stem-loop structure combined with a thiolated tetrahedron DNA nanostructure at the bottom. The top portion hybridized with the lung cancer correlated microRNA, while the bottom portion was self-assembled on gold disk electrode surface, which was modified with gold nanoparticles (Au NPs) and blocked with mercaptoethanol (MCH). The prepn. process and the performance of the proposed electrochem. genosensor were characterized by SEM (SEM), at. force microscopy (AFM), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under the optimal conditions, the developed genosensor had a detection limit of 10 pM and a good linearity with microRNA concn. ranging from 100 pM to 1 μM, which showed a great potential in highly sensitive clin. cancer diagnosis application.
- 4Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630 DOI: 10.1038/nmat961Google ScholarThere is no corresponding record for this reference.
- 5Pumera, M.; Sanchez, S.; Ichinose, I.; Tang, J. Sens. Actuators, B 2007, 123, 1195 DOI: 10.1016/j.snb.2006.11.016Google Scholar5Electrochemical nanobiosensorsPumera, Martin; Sanchez, Samuel; Ichinose, Izumi; Tang, JieSensors and Actuators, B: Chemical (2007), 123 (2), 1195-1205CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)A review. This review discusses main techniques and methods which use nanoscale materials for construction of electrochem. biosensors. Described approaches include nanotube and nanoparticle-based electrodes relying on aligned nanotube arrays, direct electron transfer between biomol. and electrode, novel binding materials and mass prodn. technol.; and nanoscale materials as biomol. tracers, including gold nanoparticles, quantum dots for DNA and protein multiplexing, novel nanobiolabels as apoferritin, liposomes and enzyme tags loaded carbon nanotubes. Specific issues related to electrochem. of nanoscale materials are discussed. Various applications for genomic and proteomic anal. are described.
- 6Sheehan, P. E.; Whitman, L. J. Nano Lett. 2005, 5, 803 DOI: 10.1021/nl050298xGoogle Scholar6Detection limits for nanoscale biosensorsSheehan, Paul E.; Whitman, Lloyd J.Nano Letters (2005), 5 (4), 803-807CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)We examine through anal. calcns. and finite element simulations how the detection efficiency of disk and wire-like biosensors in unmixed fluids varies with size from the micrometer to nanometer scales. Specifically, we det. the total flux of DNA-like analyte mols. on a sensor as a function of time and flow rate for a sensor incorporated into a microfluidic system. In all cases, sensor size and shape profoundly affect the total analyte flux. The calcns. reveal that reported femtomolar detection limits for biomol. assays are very likely an analyte transport limitation, not a signal transduction limitation. We conclude that without directed transport of biomols., individual nanoscale sensors will be limited to picomolar-order sensitivity for practical time scales.
- 7Siwy, Z.; Trofin, L.; Kohli, P.; Baker, L. A.; Trautmann, C.; Martin, C. R. J. Am. Chem. Soc. 2005, 127, 5000 DOI: 10.1021/ja043910fGoogle Scholar7Protein Biosensors Based on Biofunctionalized Conical Gold NanotubesSiwy, Zuzanna; Trofin, Lacramioara; Kohli, Punit; Baker, Lane A.; Trautmann, Christina; Martin, Charles R.Journal of the American Chemical Society (2005), 127 (14), 5000-5001CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the α-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current assocd. with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biol. nanopore with an abiotic equiv. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mech. and chem. robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not obsd. Instead, the protein analyte binds to a biochem. mol.-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different mol.-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/Ig, and (iii) an antibody to the protein ricin with ricin as the analyte.
- 8Wang, J. Anal. Chim. Acta 2003, 500, 247 DOI: 10.1016/S0003-2670(03)00725-6Google ScholarThere is no corresponding record for this reference.
- 9Wang, J. Small 2005, 1, 1036 DOI: 10.1002/smll.200500214Google ScholarThere is no corresponding record for this reference.
- 10Wang, J. Analyst 2005, 130, 421 DOI: 10.1039/b414248aGoogle Scholar10Nanomaterial-based electrochemical biosensorsWang, JosephAnalyst (Cambridge, United Kingdom) (2005), 130 (4), 421-426CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)A review. The unique properties of nanoscale materials offer excellent prospects for interfacing biol. recognition events with electronic signal transduction and for designing a new generation of bioelectronic devices exhibiting novel functions. In this Highlight recent research that has led to powerful nanomaterial-based elec. biosensing devices and examine future prospects and challenges are addressed. New nanoparticle-based signal amplification and coding strategies for bioaffinity assays are discussed, along with carbon-nanotube mol. wires for achieving efficient elec. communication with redox enzyme and nanowire-based label-free DNA sensors.
- 11Seeman, N. C. J. Theor. Biol. 1982, 99, 237 DOI: 10.1016/0022-5193(82)90002-9Google Scholar11Nucleic acid junctions and latticesSeeman, Nadrian C.Journal of Theoretical Biology (1982), 99 (2), 237-47CODEN: JTBIAP; ISSN:0022-5193.It is possible to generate sequences of oligomeric nucleic acids which will preferentially assoc. to form migrationally immobile junctions, rather than linear duplexes. These structures are predicted on the maximization of Watson-Crick base pairing and the lack of sequence symmetry customarily found in their analogs in living systems. Criteria which oligonucleotide sequences must fulfill to yield these junction structures are presented. The generable junctions are nexuses, from which 3-8 double helices may emanate. Each junction may be treated as a macromol. valence cluster, and individual clusters may be linked together directly, or with pieces of linear DNA interspersed between them. This covalent linkage can be done with enormous specificity, using sticky-ended ligation techniques. It appears to be possible to generate covalently joined 3-dimensional networks of nucleic acids which are periodic in connectivity and perhaps in space.
- 12Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1 DOI: 10.1088/0957-0233/15/2/R01Google Scholar12Recent trends in electrochemical DNA biosensor technologyKerman, Kagan; Kobayashi, Masaaki; Tamiya, EiichiMeasurement Science and Technology (2004), 15 (2), R1-R11CODEN: MSTCEP; ISSN:0957-0233. (Institute of Physics Publishing)A review. Recent trends and challenges in the electrochem. methods for the detection of DNA hybridization are reviewed. Electrochem. has superior properties over the other existing measurement systems, because electrochem. biosensors can provide rapid, simple and low-cost on-field detection. Electrochem. measurement protocols are also suitable for mass fabrication of miniaturized devices. Electrochem. detection of hybridization is mainly based on the differences in the electrochem. behavior of the labels towards the hybridization reaction on the electrode surface or in the soln. Basic criteria for electrochem. DNA biosensor technol., and already commercialized products, are also introduced. Future prospects towards PCR-free DNA chips are discussed.
- 13Brown, C. W., 3rd; Buckhout-White, S.; Diaz, S. A.; Melinger, J. S.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. ACS Sens. 2017, 2, 401 DOI: 10.1021/acssensors.6b00778Google Scholar13Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular BiosensingBrown, Carl W.; Buckhout-White, Susan; Diaz, Sebastian A.; Melinger, Joseph S.; Ancona, Mario G.; Goldman, Ellen R.; Medintz, Igor L.ACS Sensors (2017), 2 (3), 401-410CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochem. inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, the authors study four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral sepn. between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism are considered. The performance of these mechanisms, their ability to reset, and how they might be used in biosensing applications are discussed.
- 14Li, D.; Song, S. P.; Fan, C. H. Acc. Chem. Res. 2010, 43, 631 DOI: 10.1021/ar900245uGoogle ScholarThere is no corresponding record for this reference.
- 15Zhao, M. Z.; Wang, X.; Ren, S. K.; Xing, Y. K.; Wang, J.; Teng, N.; Zhao, D. X.; Liu, W.; Zhu, D.; Su, S.; Sho, J. Y.; Song, S.; Wang, L. H.; Chao, J.; Wang, L. H. ACS Appl. Mater. Interfaces 2017, 9, 21942 DOI: 10.1021/acsami.7b05959Google ScholarThere is no corresponding record for this reference.
- 16Dietz, H.; Douglas, S. M.; Shih, W. M. Science 2009, 325, 725 DOI: 10.1126/science.1174251Google Scholar16Folding DNA into Twisted and Curved Nanoscale ShapesDietz, Hendrik; Douglas, Shawn M.; Shih, William M.Science (Washington, DC, United States) (2009), 325 (5941), 725-730CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly crosslinked double helixes, arrayed in parallel to their helical axes. Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quant. controlled, and a radius of curvature as tight as 6 nm was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.
- 17Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. Nature 2009, 459, 414 DOI: 10.1038/nature08016Google Scholar17Self-assembly of DNA into nanoscale three-dimensional shapesDouglas, Shawn M.; Dietz, Hendrik; Liedl, Tim; Hogberg, Bjorn; Graf, Franziska; Shih, William M.Nature (London, United Kingdom) (2009), 459 (7245), 414-418CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase scaffold strand' that is folded into a flat array of antiparallel helixes by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helixes constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concns. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manuf. of sophisticated devices bearing features on the nanometer scale.
- 18He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. D. Nature 2008, 452, 198 DOI: 10.1038/nature06597Google Scholar18Hierarchical self-assembly of DNA into symmetric supramolecular polyhedraHe, Yu; Ye, Tao; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, ChengdeNature (London, United Kingdom) (2008), 452 (7184), 198-201CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute mol. computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large nos. (hundreds) of unique DNA strands poses a challenging design problem. Here, the authors demonstrate a simple soln. to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger 3-dimensional structures. The authors test this hierarchical self-assembly concept with DNA mols. that form 3-point-star motifs, or tiles. By controlling the flexibility and concn. of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometers in size and comprised of four, twenty or sixty individual tiles, resp. The authors expect that this assembly strategy can be adapted to allow the fabrication of a range of relatively complex 3-dimensional structures.
- 19Rothemund, P. W. K. Nature 2006, 440, 297 DOI: 10.1038/nature04586Google Scholar19Folding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 20Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585 DOI: 10.1126/science.1132493Google Scholar20Enzyme-Free Nucleic Acid Logic CircuitsSeelig, Georg; Soloveichik, David; Zhang, David Yu; Winfree, ErikScience (Washington, DC, United States) (2006), 314 (5805), 1585-1588CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Biol. organisms perform complex information processing and control tasks using sophisticated biochem. circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, elec. engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degrdn. The authors report the design and exptl. implementation of DNA-based digital logic circuits. The authors demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biol. nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnol. and bioengineering.
- 21Dirks, R. M.; Pierce, N. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15275 DOI: 10.1073/pnas.0407024101Google Scholar21Triggered amplification by hybridization chain reactionDirks, Robert M.; Pierce, Niles A.Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (43), 15275-15278CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We introduce the concept of hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment. In the simplest version of this process, two stable species of DNA hairpins coexist in soln. until the introduction of initiator strands triggers a cascade of hybridization events that yields nicked double helixes analogous to alternating copolymers. The av. mol. wt. of the HCR products varies inversely with initiator concn. Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications.
- 22Yurke, B.; Turberfield, A. J.; Mills, A. P.; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605 DOI: 10.1038/35020524Google Scholar22A DNA-fuelled molecular machine made of DNAYurke, Bernard; Turberfield, Andrew J.; Mills, Allen P., Jr.; Simmel, Friedrich C.; Neumann, Jennifer L.Nature (London) (2000), 406 (6796), 605-608CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. recognition between complementary strands of DNA allows construction on a nanometer length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semi-conductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible - for example, a nanomech. switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chem. modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addn. of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.
- 23Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F.; Gothelf, K. V.; Kjems, J. Nature 2009, 459, 73 DOI: 10.1038/nature07971Google ScholarThere is no corresponding record for this reference.
- 24Nutiu, R.; Li, Y. F. J. Am. Chem. Soc. 2003, 125, 4771 DOI: 10.1021/ja028962oGoogle ScholarThere is no corresponding record for this reference.
- 25Liu, J. W.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90 DOI: 10.1002/anie.200502589Google Scholar25Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticlesLiu, Juewen; Lu, YiAngewandte Chemie, International Edition (2006), 45 (1), 90-94CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA aptamers have been used to assemble DNA-functionalized gold nanoparticles to produce highly sensitive and selective colorimetric sensors with an instantaneous color response on addn. of a substrate. The general method has been shown for adenosine and cocaine, but should be applicable to any aptamer of choice.
- 26Wang, F.; Lu, C.-H.; Willner, I. Chem. Rev. 2014, 114, 2881 DOI: 10.1021/cr400354zGoogle Scholar26From Cascaded Catalytic Nucleic Acids to Enzyme-DNA Nanostructures: Controlling Reactivity, Sensing, Logic Operations, and Assembly of Complex StructuresWang, Fuan; Lu, Chun-Hua; Willner, ItamarChemical Reviews (Washington, DC, United States) (2014), 114 (5), 2881-2941CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review on different recent approaches to tailor "smart" DNA nanostructures for autonomous activation of catalytic DNA cascades and their use for sensing, logic operations, and assembly of complex nanostructures. Topics included are enzyme-free nucleic acid-activated chain reactions, DNAzyme-activated chain reactions, enzyme/DNAzyme coupled catalytic cascades, and enzyme-nucleic acid systems for controlled chem. processes.
- 27Brown, C. W., 3rd; Lakin, M. R.; Horwitz, E. K.; Fanning, M. L.; West, H. E.; Stefanovic, D.; Graves, S. W. Angew. Chem., Int. Ed. 2014, 53, 7183 DOI: 10.1002/anie.201402691Google ScholarThere is no corresponding record for this reference.
- 28Liu, J.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 9838 DOI: 10.1021/ja0717358Google Scholar28A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivityLiu, Juewen; Lu, YiJournal of the American Chemical Society (2007), 129 (32), 9838-9839CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Copper is a key metal ion both in environment monitoring and in biol., and exposure to high concn. of copper can cause adverse health effects. Although significant progresses were made in designing fluorescent sensors for diamagnetic metal ions, few effective Cu2+ sensors are known because of the paramagnetic nature of the metal ion. The authors herein report a highly sensitive and selective turn-on fluorescent Cu2+ sensor based on an in vitro selected DNAzyme (also known as catalytic DNA or deoxyribozyme). The substrate strand of the DNAzyme was labeled with a fluorophore on the 3'-end and a quencher on the 5'-end, and the enzyme strand was labeled with a second quencher on the 5'-end. Initially, the fluorescence was quenched. Addn. of Cu2+ induced oxidative cleavage of the substrate, and the fluorescence intensity increased by 13-fold. The sensor has a detection limit of 35 nM and a dynamic range up to 20 μM. The sensor selectivity is >2000-fold for Cu2+ over Fe2+ and UO22+ and >10,000-fold over any other metal ions. The DNAzyme catalytic beacon method demonstrated here can be applied to designing turn-on fluorescent sensors for other paramagnetic metal ions.
- 29Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587 DOI: 10.1002/anie.200702006Google Scholar29Rational design of "turn-on" allosteric DNAzyme catalytic beacons for aqueous mercury ions with ultrahigh sensitivity and selectivityLiu, Juewen; Lu, YiAngewandte Chemie, International Edition (2007), 46 (40), 7587-7590CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors designed rationally highly sensitive and selective beacon for Hg based on a uranium-specific DNAzyme. Hg2+ ions enhanced the DNAzyme activity through allosteric interactions, and a series of allosteric DNAzymes with a varying no. of thymine-thymine mismatches were tested. The optimal DNAzyme was labeled with fluorophores and quenchers to construct a catalytic beacon. The sensor has a detection limit of 2.4 nM, which is lower than the EPA limit of Hg2+ ions in drinking water. It is also highly selective and is silent to any other metal ions with up to millimolar concn. levels. The catalytic-beacon performance may be further improved by the incorporation of in vitro selections to optimize the allosteric interactions. This work further demonstrated that DNAzymes are a great platform for metal sensing.
- 30Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642 DOI: 10.1021/ja034775uGoogle Scholar30A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of Gold NanoparticlesLiu, Juewen; Lu, YiJournal of the American Chemical Society (2003), 125 (22), 6642-6643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5'-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The detection level can be tuned to several orders of magnitude, from 100 nM to over 200 μM, through addn. of an inactive variant of the DNAzyme. The concept developed here can be applied to the design of nucleic acid enzyme/nanoparticle sensors for analytes that are subject to in vitro selection, and thus can significantly expand the scope of nanomaterial applications and provide a novel approach to designing simple colorimetric biosensors.
- 31Willner, I.; Shlyahovsky, B.; Zayats, M.; Willner, B. Chem. Soc. Rev. 2008, 37, 1153 DOI: 10.1039/b718428jGoogle Scholar31DNAzymes for sensing, nanobiotechnology and logic gate applicationsWillner, Itamar; Shlyahovsky, Bella; Zayats, Maya; Willner, BilhaChemical Society Reviews (2008), 37 (6), 1153-1165CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.
- 32Zuo, X. L.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816 DOI: 10.1021/ja909551bGoogle Scholar32Sensitive and selective amplified fluorescence DNA detection based on exonuclease III-aided target recyclingZuo, Xiaolei; Xia, Fan; Xiao, Yi; Plaxco, Kevin W.Journal of the American Chemical Society (2010), 132 (6), 1816-1818CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as mol. beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here the authors demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target mols., thus leading to greatly improved sensitivity relative to, for example, traditional mol. beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concns. as low as 10 pM when performed at 37°, which represents a significant improvement over the equiv. mol. beacon alone. Moreover, at 4° a detection limit as low as 20 aM could be obtained, albeit at the cost of a 24 h incubation period. Finally, this assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.
- 33Loenen, W. A. M.; Dryden, D. T. F.; Raleigh, E. A.; Wilson, G. G.; Murray, N. E. Nucleic Acids Res. 2014, 42, 3 DOI: 10.1093/nar/gkt990Google Scholar33Highlights of the DNA cutters: a short history of the restriction enzymesLoenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E.Nucleic Acids Research (2014), 42 (1), 3-19CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technol. that has transformed mol. biol. and medicine. This review traces the discovery of restriction enzymes and their continuing impact on mol. biol. and medicine.
- 34Pingoud, A.; Wilson, G. G.; Wende, W. Nucleic Acids Res. 2014, 42, 7489 DOI: 10.1093/nar/gku447Google ScholarThere is no corresponding record for this reference.
- 35Roberts, R. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5905 DOI: 10.1073/pnas.0500923102Google ScholarThere is no corresponding record for this reference.
- 36Buckhout-White, S.; Claussen, J. C.; Melinger, J. S.; Dunningham, Z.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. RSC Adv. 2014, 4, 48860 DOI: 10.1039/C4RA10580JGoogle Scholar36A triangular three-dye DNA switch capable of reconfigurable molecular logicBuckhout-White, Susan; Claussen, Jonathan C.; Melinger, Joseph S.; Dunningham, Zaire; Ancona, Mario G.; Goldman, Ellen R.; Medintz, Igor L.RSC Advances (2014), 4 (90), 48860-48871CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Structural DNA nanotechnol. has developed profoundly in the last several years allowing for the creation of increasingly sophisticated devices capable of discrete sensing, locomotion, and mol. logic. The latter research field is particularly attractive as it provides information processing capabilities that may eventually be applied in situ, for example in cells, with potential for even further coupling to an active response such as drug delivery. Rather than design a new DNA assembly for each intended logic application, it would be useful to have one generalized design that could provide multiple different logic gates or states for a targeted use. In pursuit of this goal, we demonstrate a switchable, triangular dye-labeled three-arm DNA scaffold where the individual arms can be assembled in different combinations and the linkage between each arm can be phys. removed using toehold-mediated strand displacement and then replaced by a rapid anneal. Rearranging this core structure alters the rates of Forster resonance energy transfer (FRET) between each of the two or three pendant dyes giving rise to a rich library of unique spectral signatures that ultimately form the basis for mol. photonic logic gates. The DNA scaffold is designed such that different linker lengths joining each arm, and which are used as the inputs here, can also be used independently of one another thus enhancing the range of mol. gates. The functionality of this platform structure is highlighted by easily configuring it into a series of one-, two- and three-input photonic Boolean logic gates such as OR, AND, INHIBIT, etc., along with a photonic keypad lock. Different gates can be realized in the same structure by altering which dyes are interrogated and implementation of toehold-mediated strand displacement and/or annealing allows reconfigurable switching between input states within a single logic gate as well as between two different gating devices.
- 37Buckhout-White, S.; Brown, C. W.; Hastman, D. A.; Ancona, M. G.; Melinger, J. S.; Goldman, E. R.; Medintz, I. L. RSC Adv. 2016, 6, 97587 DOI: 10.1039/C6RA23079BGoogle Scholar37Expanding molecular logic capabilities in DNA-scaffolded multiFRET triadsBuckhout-White, Susan; Brown III, Carl W.; Hastman, David A. Jr.; Ancona, Mario G.; Melinger, Joseph S.; Goldman, Ellen R.; Medintz, Igor L.RSC Advances (2016), 6 (100), 97587-97598CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Dynamic rearrangement of DNA nanostructures provides a straightforward yet powerful mechanism for sequence-specific sensing and potential signaling of such interactions. These rearrangements are often interpreted in the context of Boolean logic gates as a means of both reflecting the underlying sensing and providing preliminary processing of the raw data. Here, we expand on previous work to optimize both the sensing and signal transduction of an initial DNA-triad sensor prototype. The core structure of this DNA triad consists of dye-labeled arms connected by 1, 2, or 3 single-stranded DNA linkers, whose presence and length alter the efficiency of Forster resonance energy transfer (FRET) between the dyes. The latter forms the basis for sensing through the use of DNA hybridization and displacement which result in structural rearrangements with each configuration correlated to a different logic state. Three different avenues were pursued to optimize the sensor function: (1) restructuring the connecting linkers and dye-choices in the original structure; (2) changing the mechanism of distance modulation between the arms; and (3) moving the signaling dyes to within the single-stranded portion of the structure. The first approach provided for improvements in FRET properties and the ability to reconfigure and switch the sensors between different types of Boolean logic gates such as going from INHIBIT 1 to Enabled OR by changing dyes, for example. The last approach proved to be the most versatile providing for the largest changes in FRET along with the ability to be repeatedly toggled and reset for multiple sequential sensing events. Switching could be completed in an isothermal manner with a near stoichiometric concn. of inputs and input complements. The continued development and potential applications of these and similar types of DNA sensors are discussed.
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Abstract
Figure 1
Figure 1. (A) Schematic of the DNA switch structure and mechanism for rearrangement via enzymatic cleavage: (i) partial structure schematic indicating the arms and cleavage site. Each of these structures contains two molecular dyes, and the cleavage is transduced by simple FRET behavior; (ii) full three-arm structure, which contains three dyes and is transduced through examining each of the three acceptor-to-donor ratios. The linker between each arm of the structure is a 10 base double-stranded DNA and is coded to be a cleavage site for a specified restriction enzyme. The cleavage of one or more of these linker regions allows for the separation of the fluorescent dyes, which reduced the output of the donor-to-acceptor ratio. (B) Sequence of one of three linker sequences with the restriction site in bold and the cutting location indicated by the triangles. (C) Emission and excitation profiles of the Cy3, Cy3.5, and Cy5 molecular dyes. (D) Spectral overlap of these dyes form the basis of the multi-FRET-based optical output.
Figure 2
Figure 2. Data from single-enzyme partial structures. (A) Ratios of the acceptor dye over the donor dye photoluminescence (PL) peak values. The three plots shown each represent a different programmed sequence designed for the enzyme specified. Each sequence was tested against six different enzymes with a negative, no enzyme, and positive, no linker, control. (B) Gel electropherogram on the partial structure before and after introduction of the specific enzyme.
Figure 3
Figure 3. Assay of kinetic rate of cleavage for the partial structure cleavage. The top line in each plot represents the average of the positive control, and the bottom line is the average of the negative control.
Figure 4
Figure 4. Plots of the three acceptor-to-donor ratios for each of the full three-arm structures. (A) Single-, double-, and triple-enzyme digest for the three-arm structures. (B) Comparable control structures in which a linker is removed where the corresponding enzyme structure would be cleaved.
Figure 5
Figure 5. (A) Schematic illustration of the partial structure enzyme cleavage and rearrangement via addition of an additional Cy5-containing DNA oligo. (B) PL Intensity of each of the structures depicted in (A), with XhoI + oligo and XhoI + bridge both being one-pot reactions. (C) PL peak height plots for each of the three dyes in the system. The addition was performed both as a one-pot reaction and a sequential reaction. The controls of the switch plus the oligo or bridge are done in the absence of enzyme and show little to no reaction without the presence of the enzyme.
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- 11Seeman, N. C. J. Theor. Biol. 1982, 99, 237 DOI: 10.1016/0022-5193(82)90002-911Nucleic acid junctions and latticesSeeman, Nadrian C.Journal of Theoretical Biology (1982), 99 (2), 237-47CODEN: JTBIAP; ISSN:0022-5193.It is possible to generate sequences of oligomeric nucleic acids which will preferentially assoc. to form migrationally immobile junctions, rather than linear duplexes. These structures are predicted on the maximization of Watson-Crick base pairing and the lack of sequence symmetry customarily found in their analogs in living systems. Criteria which oligonucleotide sequences must fulfill to yield these junction structures are presented. The generable junctions are nexuses, from which 3-8 double helices may emanate. Each junction may be treated as a macromol. valence cluster, and individual clusters may be linked together directly, or with pieces of linear DNA interspersed between them. This covalent linkage can be done with enormous specificity, using sticky-ended ligation techniques. It appears to be possible to generate covalently joined 3-dimensional networks of nucleic acids which are periodic in connectivity and perhaps in space.
- 12Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1 DOI: 10.1088/0957-0233/15/2/R0112Recent trends in electrochemical DNA biosensor technologyKerman, Kagan; Kobayashi, Masaaki; Tamiya, EiichiMeasurement Science and Technology (2004), 15 (2), R1-R11CODEN: MSTCEP; ISSN:0957-0233. (Institute of Physics Publishing)A review. Recent trends and challenges in the electrochem. methods for the detection of DNA hybridization are reviewed. Electrochem. has superior properties over the other existing measurement systems, because electrochem. biosensors can provide rapid, simple and low-cost on-field detection. Electrochem. measurement protocols are also suitable for mass fabrication of miniaturized devices. Electrochem. detection of hybridization is mainly based on the differences in the electrochem. behavior of the labels towards the hybridization reaction on the electrode surface or in the soln. Basic criteria for electrochem. DNA biosensor technol., and already commercialized products, are also introduced. Future prospects towards PCR-free DNA chips are discussed.
- 13Brown, C. W., 3rd; Buckhout-White, S.; Diaz, S. A.; Melinger, J. S.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. ACS Sens. 2017, 2, 401 DOI: 10.1021/acssensors.6b0077813Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular BiosensingBrown, Carl W.; Buckhout-White, Susan; Diaz, Sebastian A.; Melinger, Joseph S.; Ancona, Mario G.; Goldman, Ellen R.; Medintz, Igor L.ACS Sensors (2017), 2 (3), 401-410CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochem. inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, the authors study four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral sepn. between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism are considered. The performance of these mechanisms, their ability to reset, and how they might be used in biosensing applications are discussed.
- 14Li, D.; Song, S. P.; Fan, C. H. Acc. Chem. Res. 2010, 43, 631 DOI: 10.1021/ar900245uThere is no corresponding record for this reference.
- 15Zhao, M. Z.; Wang, X.; Ren, S. K.; Xing, Y. K.; Wang, J.; Teng, N.; Zhao, D. X.; Liu, W.; Zhu, D.; Su, S.; Sho, J. Y.; Song, S.; Wang, L. H.; Chao, J.; Wang, L. H. ACS Appl. Mater. Interfaces 2017, 9, 21942 DOI: 10.1021/acsami.7b05959There is no corresponding record for this reference.
- 16Dietz, H.; Douglas, S. M.; Shih, W. M. Science 2009, 325, 725 DOI: 10.1126/science.117425116Folding DNA into Twisted and Curved Nanoscale ShapesDietz, Hendrik; Douglas, Shawn M.; Shih, William M.Science (Washington, DC, United States) (2009), 325 (5941), 725-730CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly crosslinked double helixes, arrayed in parallel to their helical axes. Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quant. controlled, and a radius of curvature as tight as 6 nm was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.
- 17Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. Nature 2009, 459, 414 DOI: 10.1038/nature0801617Self-assembly of DNA into nanoscale three-dimensional shapesDouglas, Shawn M.; Dietz, Hendrik; Liedl, Tim; Hogberg, Bjorn; Graf, Franziska; Shih, William M.Nature (London, United Kingdom) (2009), 459 (7245), 414-418CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase scaffold strand' that is folded into a flat array of antiparallel helixes by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helixes constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concns. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manuf. of sophisticated devices bearing features on the nanometer scale.
- 18He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. D. Nature 2008, 452, 198 DOI: 10.1038/nature0659718Hierarchical self-assembly of DNA into symmetric supramolecular polyhedraHe, Yu; Ye, Tao; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, ChengdeNature (London, United Kingdom) (2008), 452 (7184), 198-201CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute mol. computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large nos. (hundreds) of unique DNA strands poses a challenging design problem. Here, the authors demonstrate a simple soln. to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger 3-dimensional structures. The authors test this hierarchical self-assembly concept with DNA mols. that form 3-point-star motifs, or tiles. By controlling the flexibility and concn. of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometers in size and comprised of four, twenty or sixty individual tiles, resp. The authors expect that this assembly strategy can be adapted to allow the fabrication of a range of relatively complex 3-dimensional structures.
- 19Rothemund, P. W. K. Nature 2006, 440, 297 DOI: 10.1038/nature0458619Folding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 20Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585 DOI: 10.1126/science.113249320Enzyme-Free Nucleic Acid Logic CircuitsSeelig, Georg; Soloveichik, David; Zhang, David Yu; Winfree, ErikScience (Washington, DC, United States) (2006), 314 (5805), 1585-1588CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Biol. organisms perform complex information processing and control tasks using sophisticated biochem. circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, elec. engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degrdn. The authors report the design and exptl. implementation of DNA-based digital logic circuits. The authors demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biol. nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnol. and bioengineering.
- 21Dirks, R. M.; Pierce, N. A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15275 DOI: 10.1073/pnas.040702410121Triggered amplification by hybridization chain reactionDirks, Robert M.; Pierce, Niles A.Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (43), 15275-15278CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)We introduce the concept of hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment. In the simplest version of this process, two stable species of DNA hairpins coexist in soln. until the introduction of initiator strands triggers a cascade of hybridization events that yields nicked double helixes analogous to alternating copolymers. The av. mol. wt. of the HCR products varies inversely with initiator concn. Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications.
- 22Yurke, B.; Turberfield, A. J.; Mills, A. P.; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605 DOI: 10.1038/3502052422A DNA-fuelled molecular machine made of DNAYurke, Bernard; Turberfield, Andrew J.; Mills, Allen P., Jr.; Simmel, Friedrich C.; Neumann, Jennifer L.Nature (London) (2000), 406 (6796), 605-608CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. recognition between complementary strands of DNA allows construction on a nanometer length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semi-conductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible - for example, a nanomech. switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chem. modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addn. of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.
- 23Andersen, E. S.; Dong, M.; Nielsen, M. M.; Jahn, K.; Subramani, R.; Mamdouh, W.; Golas, M. M.; Sander, B.; Stark, H.; Oliveira, C. L. P.; Pedersen, J. S.; Birkedal, V.; Besenbacher, F.; Gothelf, K. V.; Kjems, J. Nature 2009, 459, 73 DOI: 10.1038/nature07971There is no corresponding record for this reference.
- 24Nutiu, R.; Li, Y. F. J. Am. Chem. Soc. 2003, 125, 4771 DOI: 10.1021/ja028962oThere is no corresponding record for this reference.
- 25Liu, J. W.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90 DOI: 10.1002/anie.20050258925Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticlesLiu, Juewen; Lu, YiAngewandte Chemie, International Edition (2006), 45 (1), 90-94CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA aptamers have been used to assemble DNA-functionalized gold nanoparticles to produce highly sensitive and selective colorimetric sensors with an instantaneous color response on addn. of a substrate. The general method has been shown for adenosine and cocaine, but should be applicable to any aptamer of choice.
- 26Wang, F.; Lu, C.-H.; Willner, I. Chem. Rev. 2014, 114, 2881 DOI: 10.1021/cr400354z26From Cascaded Catalytic Nucleic Acids to Enzyme-DNA Nanostructures: Controlling Reactivity, Sensing, Logic Operations, and Assembly of Complex StructuresWang, Fuan; Lu, Chun-Hua; Willner, ItamarChemical Reviews (Washington, DC, United States) (2014), 114 (5), 2881-2941CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review on different recent approaches to tailor "smart" DNA nanostructures for autonomous activation of catalytic DNA cascades and their use for sensing, logic operations, and assembly of complex nanostructures. Topics included are enzyme-free nucleic acid-activated chain reactions, DNAzyme-activated chain reactions, enzyme/DNAzyme coupled catalytic cascades, and enzyme-nucleic acid systems for controlled chem. processes.
- 27Brown, C. W., 3rd; Lakin, M. R.; Horwitz, E. K.; Fanning, M. L.; West, H. E.; Stefanovic, D.; Graves, S. W. Angew. Chem., Int. Ed. 2014, 53, 7183 DOI: 10.1002/anie.201402691There is no corresponding record for this reference.
- 28Liu, J.; Lu, Y. J. Am. Chem. Soc. 2007, 129, 9838 DOI: 10.1021/ja071735828A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivityLiu, Juewen; Lu, YiJournal of the American Chemical Society (2007), 129 (32), 9838-9839CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Copper is a key metal ion both in environment monitoring and in biol., and exposure to high concn. of copper can cause adverse health effects. Although significant progresses were made in designing fluorescent sensors for diamagnetic metal ions, few effective Cu2+ sensors are known because of the paramagnetic nature of the metal ion. The authors herein report a highly sensitive and selective turn-on fluorescent Cu2+ sensor based on an in vitro selected DNAzyme (also known as catalytic DNA or deoxyribozyme). The substrate strand of the DNAzyme was labeled with a fluorophore on the 3'-end and a quencher on the 5'-end, and the enzyme strand was labeled with a second quencher on the 5'-end. Initially, the fluorescence was quenched. Addn. of Cu2+ induced oxidative cleavage of the substrate, and the fluorescence intensity increased by 13-fold. The sensor has a detection limit of 35 nM and a dynamic range up to 20 μM. The sensor selectivity is >2000-fold for Cu2+ over Fe2+ and UO22+ and >10,000-fold over any other metal ions. The DNAzyme catalytic beacon method demonstrated here can be applied to designing turn-on fluorescent sensors for other paramagnetic metal ions.
- 29Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 7587 DOI: 10.1002/anie.20070200629Rational design of "turn-on" allosteric DNAzyme catalytic beacons for aqueous mercury ions with ultrahigh sensitivity and selectivityLiu, Juewen; Lu, YiAngewandte Chemie, International Edition (2007), 46 (40), 7587-7590CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors designed rationally highly sensitive and selective beacon for Hg based on a uranium-specific DNAzyme. Hg2+ ions enhanced the DNAzyme activity through allosteric interactions, and a series of allosteric DNAzymes with a varying no. of thymine-thymine mismatches were tested. The optimal DNAzyme was labeled with fluorophores and quenchers to construct a catalytic beacon. The sensor has a detection limit of 2.4 nM, which is lower than the EPA limit of Hg2+ ions in drinking water. It is also highly selective and is silent to any other metal ions with up to millimolar concn. levels. The catalytic-beacon performance may be further improved by the incorporation of in vitro selections to optimize the allosteric interactions. This work further demonstrated that DNAzymes are a great platform for metal sensing.
- 30Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642 DOI: 10.1021/ja034775u30A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of Gold NanoparticlesLiu, Juewen; Lu, YiJournal of the American Chemical Society (2003), 125 (22), 6642-6643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5'-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The detection level can be tuned to several orders of magnitude, from 100 nM to over 200 μM, through addn. of an inactive variant of the DNAzyme. The concept developed here can be applied to the design of nucleic acid enzyme/nanoparticle sensors for analytes that are subject to in vitro selection, and thus can significantly expand the scope of nanomaterial applications and provide a novel approach to designing simple colorimetric biosensors.
- 31Willner, I.; Shlyahovsky, B.; Zayats, M.; Willner, B. Chem. Soc. Rev. 2008, 37, 1153 DOI: 10.1039/b718428j31DNAzymes for sensing, nanobiotechnology and logic gate applicationsWillner, Itamar; Shlyahovsky, Bella; Zayats, Maya; Willner, BilhaChemical Society Reviews (2008), 37 (6), 1153-1165CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.
- 32Zuo, X. L.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816 DOI: 10.1021/ja909551b32Sensitive and selective amplified fluorescence DNA detection based on exonuclease III-aided target recyclingZuo, Xiaolei; Xia, Fan; Xiao, Yi; Plaxco, Kevin W.Journal of the American Chemical Society (2010), 132 (6), 1816-1818CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as mol. beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here the authors demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target mols., thus leading to greatly improved sensitivity relative to, for example, traditional mol. beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concns. as low as 10 pM when performed at 37°, which represents a significant improvement over the equiv. mol. beacon alone. Moreover, at 4° a detection limit as low as 20 aM could be obtained, albeit at the cost of a 24 h incubation period. Finally, this assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.
- 33Loenen, W. A. M.; Dryden, D. T. F.; Raleigh, E. A.; Wilson, G. G.; Murray, N. E. Nucleic Acids Res. 2014, 42, 3 DOI: 10.1093/nar/gkt99033Highlights of the DNA cutters: a short history of the restriction enzymesLoenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E.Nucleic Acids Research (2014), 42 (1), 3-19CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)A review. In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technol. that has transformed mol. biol. and medicine. This review traces the discovery of restriction enzymes and their continuing impact on mol. biol. and medicine.
- 34Pingoud, A.; Wilson, G. G.; Wende, W. Nucleic Acids Res. 2014, 42, 7489 DOI: 10.1093/nar/gku447There is no corresponding record for this reference.
- 35Roberts, R. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5905 DOI: 10.1073/pnas.0500923102There is no corresponding record for this reference.
- 36Buckhout-White, S.; Claussen, J. C.; Melinger, J. S.; Dunningham, Z.; Ancona, M. G.; Goldman, E. R.; Medintz, I. L. RSC Adv. 2014, 4, 48860 DOI: 10.1039/C4RA10580J36A triangular three-dye DNA switch capable of reconfigurable molecular logicBuckhout-White, Susan; Claussen, Jonathan C.; Melinger, Joseph S.; Dunningham, Zaire; Ancona, Mario G.; Goldman, Ellen R.; Medintz, Igor L.RSC Advances (2014), 4 (90), 48860-48871CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Structural DNA nanotechnol. has developed profoundly in the last several years allowing for the creation of increasingly sophisticated devices capable of discrete sensing, locomotion, and mol. logic. The latter research field is particularly attractive as it provides information processing capabilities that may eventually be applied in situ, for example in cells, with potential for even further coupling to an active response such as drug delivery. Rather than design a new DNA assembly for each intended logic application, it would be useful to have one generalized design that could provide multiple different logic gates or states for a targeted use. In pursuit of this goal, we demonstrate a switchable, triangular dye-labeled three-arm DNA scaffold where the individual arms can be assembled in different combinations and the linkage between each arm can be phys. removed using toehold-mediated strand displacement and then replaced by a rapid anneal. Rearranging this core structure alters the rates of Forster resonance energy transfer (FRET) between each of the two or three pendant dyes giving rise to a rich library of unique spectral signatures that ultimately form the basis for mol. photonic logic gates. The DNA scaffold is designed such that different linker lengths joining each arm, and which are used as the inputs here, can also be used independently of one another thus enhancing the range of mol. gates. The functionality of this platform structure is highlighted by easily configuring it into a series of one-, two- and three-input photonic Boolean logic gates such as OR, AND, INHIBIT, etc., along with a photonic keypad lock. Different gates can be realized in the same structure by altering which dyes are interrogated and implementation of toehold-mediated strand displacement and/or annealing allows reconfigurable switching between input states within a single logic gate as well as between two different gating devices.
- 37Buckhout-White, S.; Brown, C. W.; Hastman, D. A.; Ancona, M. G.; Melinger, J. S.; Goldman, E. R.; Medintz, I. L. RSC Adv. 2016, 6, 97587 DOI: 10.1039/C6RA23079B37Expanding molecular logic capabilities in DNA-scaffolded multiFRET triadsBuckhout-White, Susan; Brown III, Carl W.; Hastman, David A. Jr.; Ancona, Mario G.; Melinger, Joseph S.; Goldman, Ellen R.; Medintz, Igor L.RSC Advances (2016), 6 (100), 97587-97598CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)Dynamic rearrangement of DNA nanostructures provides a straightforward yet powerful mechanism for sequence-specific sensing and potential signaling of such interactions. These rearrangements are often interpreted in the context of Boolean logic gates as a means of both reflecting the underlying sensing and providing preliminary processing of the raw data. Here, we expand on previous work to optimize both the sensing and signal transduction of an initial DNA-triad sensor prototype. The core structure of this DNA triad consists of dye-labeled arms connected by 1, 2, or 3 single-stranded DNA linkers, whose presence and length alter the efficiency of Forster resonance energy transfer (FRET) between the dyes. The latter forms the basis for sensing through the use of DNA hybridization and displacement which result in structural rearrangements with each configuration correlated to a different logic state. Three different avenues were pursued to optimize the sensor function: (1) restructuring the connecting linkers and dye-choices in the original structure; (2) changing the mechanism of distance modulation between the arms; and (3) moving the signaling dyes to within the single-stranded portion of the structure. The first approach provided for improvements in FRET properties and the ability to reconfigure and switch the sensors between different types of Boolean logic gates such as going from INHIBIT 1 to Enabled OR by changing dyes, for example. The last approach proved to be the most versatile providing for the largest changes in FRET along with the ability to be repeatedly toggled and reset for multiple sequential sensing events. Switching could be completed in an isothermal manner with a near stoichiometric concn. of inputs and input complements. The continued development and potential applications of these and similar types of DNA sensors are discussed.
- 38www.neb.com, 2017.There is no corresponding record for this reference.
- 39Maffeo, C.; Luan, B. Q.; Aksimentiev, A. Nucleic Acids Res. 2012, 40, 3812 DOI: 10.1093/nar/gkr1220There is no corresponding record for this reference.
- 40Massey, M.; Kim, H.; Conroy, E. M.; Algar, W. R. J. Phys. Chem. C 2017, 121, 13345 DOI: 10.1021/acs.jpcc.7b02739There is no corresponding record for this reference.
- 41Guo, Y. H.; Yang, K. L.; Sun, J. C.; Wu, J.; Ju, H. X. Biosens. Bioelectron. 2017, 94, 651 DOI: 10.1016/j.bios.2017.03.066There is no corresponding record for this reference.
- 42Kotani, S.; Hughes, W. L. J. Am. Chem. Soc. 2017, 139, 6363 DOI: 10.1021/jacs.7b00530There is no corresponding record for this reference.
- 43Wang, B.; Wang, X. J.; Wei, B.; Huang, F. J.; Yao, D. B.; Liang, H. J. Nanoscale 2017, 9, 2981 DOI: 10.1039/C7NR00386BThere is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01333.
DNA sequences, comparative buffer analysis, full enzyme analysis, enzyme triad table, and ternary plot for three dye assemblies (PDF)
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