Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling

LNA-porphyrin building blocks were used to create stable zipper-porphyrin DNA arrays, which were analysed in detail with CD spectroscopy and thermodynamic studies.


General
Chemicals were supplied by Sigma-Aldrich, Link Technologies, Glen Research, and Berry and Associates and used as received. DNA purification columns were supplied by Glen Research and Berry and Associates. Column chromatography was carried out using silica gel (Kieselgel 60), silica gel type H and/or basic alumina (50-200 μm, Brockmann activity I). TLC was carried out on Merck aluminium backed sheets of silica gel 60 F 254 or aluminium backed sheets of alumina 60 F 254 and were visualised using UV light (254 nm and 365 nm), anisaldehyde, phosphomolybdic acid (5 % in ethanol), and potassium permanganate in water.
Proton and carbon NMR spectra were recorded at 300/400 MHz and 75/100 MHz, respectively, using either a Bruker AC300 or Bruker DPX400 spectrometer. Chemical shifts are given in ppm and spectra are calibrated to the residual solvent peak. Coupling constants (J) are given in Hertz (Hz). Assignment was aided by DEPT-135, 1 H-1 H COSY, HMQC and HMBC experiments and Lorentz-Gauss resolution enhancement data reprocessing (ACD/SpecManager 12.0, ACD Labs).
UV-vis spectroscopy experiments were conducted using a Varian Cary 300 Bio spectrophotometer, and fluorescence spectroscopy experiments were conducted using a Varian Cary Eclipse fluorescence spectrophotometer, using quartz cells (supplied by Hellma and Starna) with 1 mm, 2 mm or 1 cm path lengths. The temperature of experiments was controlled using a Varian Cary Temperature Controller and peltier system with a Varian Cary Series II Temperature Probe. Concentrations of oligonucleotides (ODNs) were calculated using the Beer-Lambert law for the absorption at 260 nm. Molar extinction coefficients were obtained by the molar extinction calculator provided by IDT and by replacing the appropriate thymidines with the value of the porphyrin-dU (ε 260 = 13'460 L⋅mol −1 ⋅cm −1 ) as determined previously. [1] CD spectroscopy was recorded using a Chirascan Plus, or on Diamond Light Source beamline B23 Station B. The data was recorded in mdeg and corrected to delta epsilon using the formula Δε = θ / (10 x conc. x path length x 3298); conc. is in mol/litre, path length is in cm.
All spectra were recorded at 4 μM concentration single stranded ODN in phosphate buffer (50 mM sodium phosphate, 100 mM sodium chloride, 1 mM di-sodium EDTA, pH 7). Duplexes were formed by heating the samples to 85 °C for two minutes, followed by slow cooling (1 °C / min) to 20 °C. Stock solutions of HPLC-pure DNA were stored at 4 °C (no precipitation was observed). The ODNs were used at a concentration of 2.5 μM for melting analysis.

DNA synthesis
DNA synthesis was carried out on an Applied Biosystems Expedite machine using 500 Å pore CPG beads. DNA synthesis reagents (SAFC or Link Technologies) were used as received. Deblocking steps used 3 % TCA in DCM solution, activation steps use 0.1 M 'Activator 42' (5-(bis-3,5trifluoromethylphenyl)-1H-tetrazole) in MeCN, capping steps used acetic anhydride in THF (Cap A) and pyridine and NMI in THF (Cap B), oxidizing steps used 0.02 M iodine, pyridine and water in THF, washing steps used MeCN. Cleavage of the oligonucleotide from the solid support was achieved by passing concentrated ammonium hydroxide through the column using two syringes at either end for one hour at room temperature. This solution was then heated to 40 °C overnight to deprotect the oligonucleotide. Synthesis was conducted DMT-on. FDMT-on oligonucleotide was diluted with an equal volume of loading buffer (Berry & Associates) and purified on Fluoro-Pak TM II according to the protocol from the manufacturer. Final analysis for purity was performed using RP-HPLC which was carried out using a Varian Galaxie system using a Waters XBridge OST C18, 2.5 µm 4.6 x 50mm column. Eluents used were 8.6 mM TEA / 100 mM HFIP buffer and MeOH. Eluents were filtered through a Supelco Nylon 66 Membrane filter (0.45 µm pore size) before use. Flow rates were set to 1 mL min -1 . ε 260 Melting analysis UV-melting curves were obtained at 260 nm, 0.1 °C / min. The curves were fitted using Origin software; details can be found in the associated files available at http://dx.doi.org/10.5258/SOTON/381422. The calculation takes the thermodynamic equilibria and the slopes at the beginning and end of the melting into account, using the following equation: K eq = (exp((-H/8.831*(x+273.15))+(S/8.831))) where H = ΔH and S = ΔS, x = temperature in °C.
The absorbance y can be fitted as: y = 0.5*((1-a)*(ss*(x-T max )+2*A max )+(a*(ds*(x-T min )+2*A min ))) where ss is the slope at high temp (ssDNA) and ds is the slope at low temp (dsDNA), A max , A min , T max and T min are maximal and minimal absorbances and temperatures, respectively. a is the fraction in duplex form given as: a = (1+(1/(c*K eq ))*(1-sqrt(1+2*c*K eq ))) where c = concentration of the sample.
The equations were combined to give the fitting function as indicated in the examples below. c, R, T max and T min were held constant while all other parameters were allowed for variation until convergence of the fit.