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.     

Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.