T7 (Y639F) RNA polymerase extraction and purification to produce high yield 2?Fluoro-modified RNA aptamers through cell-SELEX procedures

MANCHA-AGRESTI, PAMELA; THIEL, WILLIAM; VARGAS, MAURICIO; SANCHEZ, DIEGO; MESTRE, MARÍA BELÉN; RUIZ CIANCIO, DARÍO; BRUNO, MARTÍN A; COTIGNOLA, JAVIER

Mar del Plata

Congreso; Reunión Anual de Biociencia 2019; 2019

Sociedad Argentina de Investigación Clínica

Nucleic acids are susceptible to ubiquitous serum nucleases. Chemical modifications to the 2? position of ribose can be used to stabilize oligonucleotides, specifically RNA aptamers. Aptamers are single stranded oligonucleotides which bind specifically to a variety of targets. The 2?-fluoro modified RNA aptamers can 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) RNA polymerase, so it could be used in a near future for the production of 2?F-modified RNA aptamers through cell-SELEX. The BL21(DE3) E. coli bacteria was used for transformation, together with the kanamycin-resistant 6xHis-Tagged T7 Y639F polymerase vector. First, chemically competent E.coli cells were produced using MgCl2 and CaCl2. Next, these competent bacteria were incubated with 100ng of the vector. At the end, the cells were streaked in LB-kanamycin resistant plate. The Y639F T7 RNAP bacteria was then inoculated with Lysozyme (10mg/mL), PMSF (20mg/mL), Leupeptin (5mg/mL), 8% Na-Deoxycholic acid and protease inhibitor cocktail. Cells were lysed and transfer to Nickel NTA magnetic agarose beads. The beads were washed to eliminate non-specifically bound protein, and then, 6xHis-Tagged T7 RNAP protein were eluted from the magnetic beads. The purified protein was used for in vitro-transcription of dsDNA template to generate an RNA aptamer. Our results demonstrated that our purified T7 polymerase could be successfully used for in vitro RNA aptamer transcription. Then, we confirmed that the dilution 1:1, and even, 2:1 of T7 RNAP are the optimal concentration for in vitro transcriptions with phenol-chloroform extraction. To conclude, it is often a challenge to get T7 polymerases that incorporate nucleotides containing modifications. Higher yields of 2?-fluoro-pyrimidine transcripts can be produced using the Y639F mutant T7 RNA polymerase purified by our group in the concentrations mentioned above.