PubMed · 8743323
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T Fitzwater, B Polisky. 1996. A SELEX primer.. https://doi.org/10.1016/s0076-6879(96)67019-0
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The enzyme avian myeloblastosis virus reverse transcriptase (AMV-RT) is routinely used for cDNA synthesis, which is generally carried out at temperatures between 37 degrees C and 42 degrees C. We show that this enzyme can support cDNA synthesis, at temperatures as high as 70 degrees C. We have utilized this property of the AMV-RT to improve the specificity of polymerase chain reaction (PCR). Furthermore, this apparently thermophilic property of the enzyme, which is an important constituent of a mesophilic organism, raises intriguing questions regarding evolution of the enzyme structure.
In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with polyadenosine sequences and to a lesser degree diadenosines and monoadenosine residues. Pausing was also inversely proportional to the potential bond strength between the nascent strand and the template at the point of termination, for both mono and dinucleotides. A run of five adenosine and four uridine residues caused most pausing on the HIV-1 template, a region which is the site of much sequence heterogeneity in HIV-1. We propose that homopolyadenosine tracts can act as termination signals for RT in the context of inverted repeats as they do for certain RNA polymerases.
Replacement of alpha-, beta- and gamma-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at alpha-, beta- and gamma-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that gamma-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the gamma-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase alpha. For dUTP analogues substituted at both gamma-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at gamma-phosphonate or C5 position. Meanwhile, C5-substituted beta, gamma-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[beta, gamma-(methylphosphinyl)methylphosphonyl]-alpha-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.