Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer.
In an effort to increase the thermostability of Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), we screened random and site-saturation libraries for variants that show increased resistance to thermal inactivation. We discovered five mutations E69K, E302R, W313F, L435G and N454K that collectively increase the half-life of MMLV RT at 55 degrees C from less than 5 min to approximately 30 min in the presence of template-primer. In addition, these mutations alter the thermal profile by increasing specific activity of the pentuple mutant (M5) over a broad range of cDNA synthesis temperatures (25-70 degrees C). We further show that M5 generates higher cDNA yields and exhibits better RT-PCR performance compared to wild-type RT when used at high temperature to amplify RNA targets containing secondary structure. Finally, we demonstrate that M5 exhibits tighter binding (lower K(m)) to template-primer, which likely protects against heat inactivation.
Arezi
Bahram
B
Agilent Technologies, Stratagene Products Division, La Jolla, CA 92037, USA. bahram.arezi@agilent.com
Hogrefe
Holly
H
D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D006358:Hot Temperature; D006801:Humans; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding; D055550:Protein Stability; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D012194:RNA-Directed DNA Polymerase