Förster Resonance Energy Transfer Nanoplatform Based on Recognition-Induced Fusion/Fission of DNA Mixed Micelles for Nucleic Acid Sensing

The dynamic nature of micellar nanostructures is employed to form a self-assembled Förster resonance energy transfer (FRET) nanoplatform for enhanced sensing of DNA. The platform consists of lipid oligonucleotide FRET probes incorporated into micellar scaffolds, where single recognition events result in fusion and fission of DNA mixed micelles, triggering the fluorescence response of multiple rather than a single FRET pair. In comparison to conventional FRET substrates where a single donor interacts with a single acceptor, the micellar multiplex FRET system showed ∼20- and ∼3-fold enhancements in the limit of detection and FRET efficiency, respectively. This supramolecular signal amplification approach could potentially be used to improve FRET-based diagnostic assays of nucleic acid and non-DNA based targets.


General materials and methods
Reagents and solvents were purchased from commercial suppliers and used without further purification, unless otherwise stated. Column chromatography was carried out using open columns packed with Merck grade 60 silica gel topped with 0.5cm of sand.
TLC analysis was performed on Merck silica gel 60 silica sheets. 1

Synthesis of bischolesterol dialcohol 3
Adapted from Kobuke 3 Bischolesterol diester 2 (558mg, 0.59mmol) was suspended in dry THF (25ml) under argon and heated to 50°C. LiBH4 (50mg, 2.30mmol) was then added, and the reaction heated to 60°C and left to stir at this temperature for 4 hours. The reaction was cooled to RT and left stirring under argon overnight. 1N aqueous HCl was added dropwise until effervescence had ceased, and the reaction mixture was diluted with water, extracted with chloroform, and dried over MgSO4. After evaporation of the solution, the residue was purified by column chromatography (4% methanol in chloroform) to yield the final product (450mg, 86%).

Synthesis of bischolesterol dibromide 4
Under an inert atmosphere, pyridine (0.2ml, 2.58mmol) was added to a solution of triphenylphosphine (1.58g, 6.01mmol) and N-bromosuccinimide (1.15g, 6.44mmol) in dry DCM (20ml). A solution of bischolesterol dialcohol 3 (950mg, 1.07mmol) in dry DCM (50ml) was then added, and the resulting reaction was left stirring under argon at RT for 3 days. The solvent was removed, co-evaporating with toluene (3 x 100ml), to give a black/brown residue which was purified by column chromatography (1:1 toluene:ethyl acetate) to yield the final product as an off white solid (603mg, 56%).

Synthesis of bischolesterol diazide 5
Adapted from Sharma and Gilmer 4 To a stirred solution of bischolesterol dibromide 4 (560mg, 0.55mmol) in anhydrous DMF (16ml) was added, sodium azide (216mg, 3.3mmol) and sodium iodide (17mg, 0.11mmol). The reaction was heated to 60°C and left to stir at this temperature under argon for 2 days. Water (50ml) was added to precipitate the final product, which was isolated by filtration and washed with water to give an off white solid (290mg, 59%).   The datasets were measured on an Agilent SuperNova diffractometer using an Atlas detector. The data collections were driven and processed, and absorption corrections was applied using CrysAlisPro. The structures of bischolesterol diester 2 and bischolesterol dibromide 4 were solved using ShelXS 5 while the structure of bischolesterol diazide 5 was solved using ShelXT. 6 All three structures were refined by a full-matrix least-squares procedure on F 2 in ShelXL. 6 All non-hydrogen atoms were refined with anisotropic displacement parameters. In bischolesterol diester 2, the hydrogen atoms bonded to N(1) and N(2) were located in the electron density and

General procedure for the synthesis of bischolesterol dye conjugates
Equimolar equivalents of bischolesterol diazide and the corresponding cyanine dye were dissolved in dry THF in an inert atmosphere. DIPEA and copper iodide were then added, and the reaction was left stirring at room temperature for 12 hours. The reaction was quenched with water, and the product extracted with DCM. Organics were combined and dried over magnesium sulphate which was subsequently removed by filtration along with undissolved copper iodide. The solvent was removed to leave crude product, which was then purified by either flash chromatography, HPLC, or both techniques. Scheme S2. Synthesis of the bischolesterol dye conjugates.

HPLC purification of the bischolesterol dye conjugates
HPLC purification of the conjugates was performed on a Kinetex C18 prep column from Phenomenex, following the method described in the table below: HPLC purification of Cy3 containing products were monitored at 260nm and 550nm whereas Cy5 containing products were monitored at 260nm and 645nm. Desired products eluted at ~25 minutes.

Synthesis and purification of oligonucleotides
Oligonucleotides were synthesised using solid phase synthesis on an Applied Biosystems ABI 394 DNA/RNA synthesiser using commercially supplied DNA synthesis grade solvents and reagents.

Standard synthesis of complementary, half-complementary, scrambled, and alkyne modified strands
Standard phosphoramidites of Bz-dA, iBu-dG, Ac-dC, and dT from Link Technologies, were added to cap unreacted material, and iodine (0.02M) in THF/pyridine/water (7:2:1) was added to oxidise the phosphotriester formed. Upon sequence completion, the resins were placed in 1ml solutions of aqueous ammonia (30%) and shaken for 3 hours to cleave strands from the resin and remove protecting groups. The solutions were then desalted with a NAP-10 column from GE Healthcare, concentrated to 1ml on a Thermo Scientific speed vac, and stored in the freezer for purification.

Purification of complementary, half-complementary, scrambled, and alkyne modified strands
Semi preparative HPLC purification was performed using a Phenomenex Clarity 5μm Oligo-RP LC 250x10mm column. 1ml of sample was injected with a run time of 45 minutes for each sample, at a flow rate of 3ml/min. The column was heated to 60°C prior to sample injection. The UV/vis absorbance of each run was monitored at 260nm. The solvent gradients used are listed in the table below: Collected fractions were evaporated to dryness, diluted to 1ml in Milli-Q water, and desalted using a NAP-10 column (GE Healthcare), whilst eluting to 1.5ml. Purity of oligonucleotides was determined by analytical HPLC using a Phenomenex Clarity 5μm Oligo RP LC 250x4.6mm column. The column was heated to 60°C prior to sample injection. 20µl of sample was injected with a run time of 45 minutes for each sample, at a flow rate of 1ml/min. Solvent gradients used were identical to semi preparative HPLC (Table S2). The UV/vis absorbance of each run was monitored at 260nm.

Purification of Cy3 and Cy5 modified control strands
Cy3/Cy5 modified strands were purified and analysed with the same columns and systems as previous strands, with the exception of a different solvent gradient being applied to account for the enhanced lipophilicity of the strands (Table S3).            The spectral overlap integral ( ) for FRET pairs (Cy3-DNA/Cy5-DNA, Cy3-bischol-DNA/Cy5-bischol-DNA-TX100) is calculated using the absorption and emission spectra of dye-DNA and dye-bischol-DNA-TX100 as shown in Figure S29. quantum yield (QY) values are obtained from the reference literature. 8,9 The corresponding Förster radius is calculated with the following formula: