Rolling Circle Transcription-Amplified Hierarchically Structured Organic–Inorganic Hybrid RNA Flowers for Enzyme Immobilization

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic–inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin–biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for β-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery.

S-3 in reaction buffer (67 mM glycine-KOH, 6.7 mM MgCl2, 10 mM β-mercaptoethanol, pH 9.5) was added to remove any free single-stranded DNA, and treated at 37 °C for 1.5 h before heat inactivation at 80 °C for 15 min. The resulting circularized template DNA was confirmed by both 15% native and denatured polyacrylamide gel electrophoresis (PAGE) analysis in TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, pH 8.3). To visualize DNA bands, the gels were stained with SYBR gold stain at room temperature for 30 min and imaged using the BioSpectrum Imaging System (Ultra-Violet Products, UK).

Synthesis of hRNFs Using RCT:
In a typical rolling circle transcription (RCT) reaction, 50 μL solution containing circular template DNA (0.6 μM), rNTP mix (2 mM), T7 RNAP (5 U/μL), and BSA (200 μg/mL) was incubated in RCT reaction buffer (40 mM Tris-HCl, 6 mM MgCl2, 2 mM spermidine, 1 mM DTT, pH 7.9) at 37 °C for 20 h. The T7 RNAP enzyme was inactivated at 65 °C for 10 min after the reaction. The final product was sonicated for 10 min, followed by washing with nuclease-free water several times to remove any free enzymes and RNA strands by centrifugation at 8,000 g for 10 min. The obtained hybrid RNA flowers (hRNFs) were redispersed in nuclease-free water and stored at 4 °C for use.

Quantification of RNA Concentration:
The RNA content in hRNFs was quantified using Quant-iT™ RiboGreen RNA assay kit. The as-synthesized hRNFs were incubated with the RiboGreen reagent in a 96-well microplate for 5 min at room temperature. The fluorescence intensity (excitation at 480 nm, emission at 520 nm) was recorded using an EnSpire® Multimode plate reader (Perkin Elmer, UK). The RNA concentration was determined from the standard curve of serial dilutions of a ribosomal RNA standard provided by the manufacturer.

Quantification of PPi Concentration:
The pyrophosphate (PPi) concentration was estimated using the PiPer pyrophosphate assay kit. The samples were mixed with a freshly prepared working solution containing Amplex Red (100 μM), pyrophosphatase (0.02 U/mL), maltose phosphorylase (4 U/μL), maltose (0.4 mM), glucose oxidase (2 U/mL), and horseradish peroxidase (0.4 U/μL), according to the manufacturer's protocols. After 2 h incubation at 37 °C, the absorbance at 565 nm was monitored using an EnSpire® Multimode plate reader (Perkin Elmer, UK) and inverted to PPi concentration using appropriate PPi standards.

Synthesis of Functional hRNFs:
For synthesis of biotin-or DBCO-functionalized hRNF (Bio-hRNFs or DBCO-hRNFs), the RCT reaction was carried out with the addition of bio-UTP or S-4 DBCO-UTP (40 μM) at 37 °C for 20 h before deactivation of T7 RNAP. For the preparation of avidin-functionalized hRNFs (Av-hRNFs), 10 μL of pre-synthesized hRNFs (RNA concentration = 50 μg/mL) was further complexed with 10 μL of avidin solution (50 μg/mL) at room temperature for at least 3 h. The resulting products were sonicated and washed with nuclease-free water three times by centrifugation at 8,000 g for 10 min. The obtained functional hRNFs (Bio-hRNFs, DBCO-hRNFs, and Av-hRNFs) were redispersed in nuclease-free water and stored at 4 °C for use. . For EDS analysis, measurements over five particles per sample were carried out. The relative atomic ratios normalized to Mg content in each particle were calculated through a standardless quantitative method with k-factors incorporated in the data processing software (Aztec Software, Oxford Instruments).
SIM Imaging: For structured illumination microscopy (SIM) imaging, fluorescently labeled-hRNF particles were prepared by additionally introducing Cy5-UTP (20 μM) to the RCT reaction mixtures, followed by the reaction at 37 °C for 20 h and purification in the same way as described above. For the imaging of QDs-labeled hRNFs, the pre-synthesized functional hRNF particles were first incubated with surface-modified QDs using a bench shaker at room temperature for 30-60 min, and then washed with nuclease-free water several times by centrifugation. 10 μL or more of the particles in nuclease-free water were mixed with 100 μL S-5 of VectaShield mounting media (Vector Laboratories) before being pipetted onto an ibidi 8well glass bottom μ-slide. The particles were left overnight in order to allow them to settle to the base of the slide by gravity. SIM imaging was performed with an Elyra PS.1 (Carl Zeiss).
Dual Beam FIB-SEM Analysis: Dual beam focused ion beam (FIB)-SEM imaging was carried out using Auriga CrossBeam Workstation (Zeiss). For imaging, samples were first fixed through resin embedding with an epoxy resin embedding kit (Sigma). A drop of sample solution in nuclease-free water was placed onto a cleaned silicon wafer chip and air-dried at room temperature. The chip was then incubated with a series of mixed ethanol and resin solution (volume ratio of ethanol to resin: 3:1, 2:1, 1:1, 1:2) for 2.5−3 h. The chip was washed with ethanol carefully to remove excess solution and air-dried between each step. The samples were left to polymerize for 48 h at 60 °C and finally coated with 10 nm chromium. A single particle was selected by SEM imaging at low magnification, followed by tilting the stage to 54°. The same particle was re-located using the FIB at a working distance of 5 mm. The sample was milled at 30 kV and 50 pA. SEM images were obtained under back-scattered electron (BSE) mode with an accelerating voltage of 1.6 keV.

Thermal Stability of STV-β-gal:
To test the thermal stability of enzyme, free STV-β-gal and STV-β-gal/Bio-hRNFs were incubated in PBS buffer at temperatures ranging from 30 to 70 °C for 10 min. After the solution was cooled down to room temperature, the RBG substrate was added, and the fluorescence intensity was measured after 15-20 min. The relative enzymatic activity was obtained by normalizing the fluorescence signals in both free STV-β-gal and STVβ-gal/Bio-hRNFs to that of free STV-β-gal or STV-β-gal/Bio-hRNFs heated at 30 °C, respectively.  The highest amounts of RNA and PPi in hRNFs with their welldefined morphology were achieved when 2 mM rNTP was used. Further increase in rNTP (4 mM) gave rise to dramatic reduction in RNA production and no appreciable particle formation. This is presumably due to the competitive interactions between free ribonucleotides and RNAP against binding to Mg 2+ ions, thus leading to a decrease in effective Mg 2+ concentration required for enzyme activity.