T7 (Y639F) RNA POLYMERASE EXTRACTION AND PURIFICATION TO PRODUCE HIGH YIELD 2?FLUORO-MODIFIED RNA APTAMERS THROUGH CELL-SELEX PROCEDURES

MANCHA AGRESTI, P.; SANCHEZ, D.; RUIZ CIANCIO, D.; BRUNO, M.A.; COTIGNOLA, J.; VARGAS, M.; COIRINI, H.; MESTRE GIMENEZ, M.B.

Mar del Plata

Congreso; Reunion Anual de Sociedades de Biociencias; 2019

Sociedad Argentina de Investigación Clínica

Nucleicacids are susceptible to ubiquitous serum nucleases. Chemical modifications tothe 2? position of ribose can be used to stabilize oligonucleotides,specifically RNA aptamers. Aptamers are single stranded oligonucleotides whichbind specifically to a variety of targets. The 2?-fluoro modified RNA aptamerscan be generated in high yield using the Y639F variant of T7 RNA polymerase.Thus, the main goal of this work is to extract and purify the T7 (Y639F) RNApolymerase, so it could be used in a near future for the production of2?F-modified RNA aptamers through cell-SELEX. The BL21(DE3) E. colibacteria was used for transformation, together with the kanamycin-resistant6xHis-Tagged T7 Y639F polymerase vector. First, chemically competent E.colicells were produced using MgCl2 and CaCl2. Next, thesecompetent bacteria were incubated with 100ng of the vector. At the end, thecells were streaked in LB-kanamycin resistant plate. The Y639F T7 RNAP bacteriawas then inoculated with Lysozyme (10mg/mL), PMSF (20mg/mL), Leupeptin(5mg/mL), 8% Na-Deoxycholic acid and protease inhibitor cocktail. Cells werelysed and transfer to Nickel NTA magnetic agarose beads. The beads were washedto eliminate non-specifically bound protein, and then, 6xHis-Tagged T7 RNAPprotein were eluted from the magnetic beads. The purified protein was used forin vitro-transcription of dsDNA template to generate an RNA aptamer. Ourresults demonstrated that our purified T7 polymerase could be successfully usedfor in vitro RNA aptamer transcription. Then, we confirmed that the dilution1:1, and even, 2:1 of T7 RNAP are the optimal concentration for in vitrotranscriptions with phenol-chloroform extraction. To conclude, it is often achallenge to get T7 polymerases that incorporate nucleotides containingmodifications. Higher yields of 2?-fluoro-pyrimidine transcripts can beproduced using the Y639F mutant T7 RNA polymerase purified by our group in theconcentrations mentioned above.