Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity.
A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3' and 5' ends and a randomized 30 nt internal sequence. The fixed sequence at the 5' end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5' overhangs with recessed 3' termini. Cleavage produced a 50 nt template and 46 nt primer with the 3' terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6-8 base G run at the 3' primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding.
DeStefano
Jeffrey J
JJ
Department of Cell Biology and Molecular Genetics, University of Maryland, Building 231, College Park, MD 20742, USA. jdestefa@umd.edu
Cristofaro
Jason V
JV
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D006147:Guanine / Q000032:analysis; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D012313:RNA / Q000737:chemistry*; D052156:SELEX Aptamer Technique; D013698:Templates, Genetic
D052157:Aptamers, Nucleotide; C034477:RNA primers; D006147:Guanine; D012313:RNA; D054303:HIV Reverse Transcriptase