Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer

Abstract

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.

Figure 1.

Structure of MMLV RT highlighting the locations of thermostable mutations. The crystal structure of MMLV RT (consisting of fingers, palm, thumb and connection domains) lacking the RNase H domain was adopted from Das and Georgiadis (24). E69 resides in the fingers, E302 and W313 in the thumb, and L435 and N454 in the connection domain.

Figure 2.

Thermal profiles. Relative RT activities were measured (Materials and Methods section) after 5 min incubations in the presence of template-primer at the temperatures indicated. Percent activity was normalized relative to the maximum activity exhibited by each RT (100%).

Figure 3.

Full-length cDNA profiling using poly(A)-tailed RNA ladder. Reactions contained 192 ng (Materials and Methods section) of each enzyme and were incubated at the indicated temperatures.

Figure 4.

CD spectra. The CD spectra of MMLV RT and M5 were measured in the absence and presence of template-primer (T:P) at 25°C (A). Melting CD spectra were measured at 212.5 nm across a range of temperatures, from 25°C to 80°C (B). Protein spectra (reported in millidegrees) were not presented in terms of molar ellipticites due to contributions from template-primer. Enzyme/template-primer spectra were not the sum of individual enzyme and template-primer spectra (indicative of conformation changes accompanying complex formation; data not shown), and thus, could not be corrected by subtracting the primer-template spectra and computing mean residue ellipiticity from calculated mean amino-acid weight.

Figure 5.

High-temperature cDNA synthesis and two-step RT–PCR. All reactions contained 384 ng RT and were incubated at indicated temperatures for 1 h, followed by 15 min at 70°C. Two microlitres of cDNA and 2.5 U of PicoMaxx were used in all amplifications. In (A) and (B), reactions also contained 500 ng human skeletal muscle total RNA. A 1.9 kb fragment of dystrophin was amplified using primers Dys8F and Dys2R. In (C), reactions contained 100 ng human hela RNA and 4 pmol Eps0.6-R. A 0.6 kb fragment of polymerase ɛ was amplified using primers Eps0.6-F and Eps0.6-R. In (D), indicated amounts of human hela total RNA were used in each reaction. RT incubation temperature was at 50°C. A 2 kb fragment of polymerase ɛ was amplified using primers Eps2-F and Eps2-R. In (E), indicated amounts of Human Universal Reference RNA were used in each reaction. RT incubation temperature was at 42°C. A 0.6-kb fragment from the 5′-end of 20-kb human nebulin transcript was amplified using primers NebF3 and NebR3.