Nucleic Acids Research Advance Access published online on December 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn952
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Nucleic Acid Enzymes |
Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer
Agilent Technologies, Stratagene Products Division, La Jolla, CA 92037, USA
*To whom correspondence should be addressed. Tel: +1 858 373 6389; Fax: +1 858 373 5300; Email: bahram.arezi{at}agilent.com
Received November 7, 2008. Revised November 7, 2008. Accepted November 11, 2008.
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°C from less than 5 min to 
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°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 Km) to template-primer, which likely protects against heat inactivation.