Reductive Charge Transfer through an RNA Aptamer

Abstract The transfer of charges through double helical DNA is a very well investigated bioelectric phenomenon. RNA, on the contrary, has been less studied in this regard. The few available data report on charge transfer through RNA duplex structures mainly composed of homonucleotide sequences. In the light of the RNA world scenarios, it is an interesting question, if charge transfer can be coupled with RNA function. Functional RNAs however, contain versatile structural motifs. Therefore, electron transport also through non‐Watson–Crick base‐paired regions might be required. We here demonstrate distance‐dependent reductive charge transfer through RNA duplexes and through the non‐Watson–Crick base‐paired region of an RNA aptamer.


Experimental Procedures
The synthesis of the 5DMAPyU phosphoramidite was described in our earlier work. [1] General Mass spectra were recorded on a Bruker microflex MALDI-TOF MS. UV/Vis absorption measurements were recorded with a JASCO V-650. Fluorescence measurements were performed with a JASCO FP-6500 spectrofluorometer. All spectra were recorded at 22 °C in Na-Pi with an RNA concentration of 2.5 μM. UV melting curves were determined using the Cary 100 spectrophotometer equipped with a temperature control unit (Varian), applying a heating/cooling rate of 0.2 °C/min in a temperature range from 10 °C or 15 °C to 90 °C. All optical spectroscopy was performed in triplet measurements, except the Tm determination of the aptamer sequences, which was done in duplicates. Na-Pi buffer was prepared to contain 20 mM sodium phosphate and 100 mM NaCl at pH 7. Phosphordiesterase (Crotalus atrox) was obtained from Sigma-Aldrich and Shrimp Alkaline Phosphatase from Affymetrix. All other reagents or chemicals and solvents were obtained as the highest commercially available grade and used without further purification.

RNA preparation
RNAs were synthesized on a Gene Assembler special DNA/RNA Synthesizer following the standard protocol for oligoribonucleotide chain assembly. Standard PAC-phosphoramidites as well as CPG supports were obtained from ChemGenes or Link Technologies. For removal of base and phosphate protecting groups and cleavage from the support, the synthesized RNAs were incubated with aqueous ammonia (32%)/ethanolic methylamine (8 M) (1:1, v/v) at 65 °C for 40 min., followed by incubation with TEA·3HF for 1.5 h at 55 °C for removal of the 2'-O-protecting groups. Purification of the synthesized RNA strands was performed by denaturing PAGE (12.5%, urea and acrylamide (acrylamide/bis-acrylamide 19:1) were purchased from Roth). Product containing bands were cut out, eluted from the gel with 0.3 M NaOAc, pH 6.5, and desalted via precipitation with ethanol. dsRNA for fluorescence and UV/Vismeasurements and for UV melting curves was prepared in Na-Pi buffer by mixing equimolar amounts of complementary strands to a final concentration of 2.5 µM. Hybridisation was promoted by heating the mixture at 90 °C for 2 min followed by slow cooling at room temperature for gradual annealing.

Irradiation of the RNA samples
The irradiation of the RNA samples was carried out using a concentration of 2.5 μM in Na-Pi buffer. The RNA solutions had been filled into quartz glass precision cuvettes (500 μl, path length 1 cm, Hellma Analytics) and were irradiated for 40 min with light of a wavelength of 365 nm (UV lamp). After irradiation, the RNA was precipitated by adding 300 vol% ethanol. Afterwards, the precipitated and dried RNA was digested enzymatically.

Enzymatic RNA digestion
The phosphodiesterase (Crotalus atrox, 0.01 U/mg) was freshly dissolved in H2O before use. The reaction mixture was prepared by mixing 6 μl Tris/HCl buffer (40 mM Tris, 40 mM MgCl2, pH 8.9) to 1 nmol of the respective RNA, followed by the addition of 3 μl of the phosphodiesterase solution (0.003 U/μl) and 1 μl of alkaline phosphatase (Shrimp Alkaline Phosphatase, 1 U/μl). The resulting reaction mixture was incubated at 37 °C for 24 h. The reaction was stopped by adding 100 μl water, followed by analysis via analytical RP-HPLC.

RP-HPLC
The digested RNA samples were run on Akta Purifier (Amersham Biosciences) using a Nucleosil 125/4 (120-5 C18) column from Macherey-Nagel with a column volume (CV) of 1.571 ml. The analytical RP-HPLC run was executed by using a flowrate of 0.5 ml/min with a solvent gradient consisting of buffer A (0.05 M TEAAc) and buffer B (0.1 M TEAAc, 30% MeCN). The used solvent gradient was: isocratic 1% (B) for 2 CV, linear gradient to 16% (B) in 12 CV, linear gradient to 100% (B) in 10 CV and isocratic 100% (B) for 4 CV.
The resulting nucleoside mixture was analyzed using nucleoside and protein standards. Inosine (I) was identified instead of adenosine, due to an adenosine deaminase-contamination of the phosphodiesterase. [2] Figure S1. UV melting curve of A3_ds and the corresponding derivation, which lacks a clear maximum for melting point determination. Figure S2. UV/Vis absorption spectra of 5DMAPyU in comparison to the 5DMAPyU modified RNA duplex sequences. All spectra were recorded at 22 °C at a concentration of 2.5 µM in Na-Pi for the RNAs and 1.5 µM for DMAPyU. The pyrene absorption range between 330 and 400 nm for each sample is magnified in the extract. Figure S3. UV/Vis absorption spectra of the 5DMAPyU modified RNA aptamer sequences. All spectra were recorded at 22 °C at a concentration of 2.5 µM in Na-Pi. The pyrene absorption range between 330 and 400 nm for each sample is magnified in the extract.