Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

Abstract

The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.Key words: dimethyl sulfoxide; formamide; glycerol; organic solvent; reverse transcriptaseReverse transcriptase (RT) [EC 2.7.7.49] is the enzyme responsible for viral genome replication. It possesses RNA-and DNA-dependent DNA polymerase and RNase H activities.1,2) RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV) are the most extensively used in cDNA synthesis 3) and RNA amplification reactions 4,5) due to their high catalytic activity and fidelity.6) The AMV RT is a heterodimer consisting of a 63-kDa subunit and a 95-kDa subunit, while MMLV RT is a 75-kDa monomer. The subunit of AMV RT comprises the fingers, palm, thumb, connection, and RNase H domains, and the subunit comprises these five domains and the C-terminal integrase domain.7) MMLV RT also contains these five domains.8) The active site of the DNA polymerase reaction resides in the fingers/palm/thumb domain, while that of the RNase H reaction lies in the RNase H domain.Enzymes, in general, are inactivated in the presence of organic solvents. However, enzymatic reactions in media containing organic additives can sometimes make previously problematic processes feasible through increased substrate solubility or diminished side reactions, for example. In polymerase chain reaction (PCR), various organic additives have been reported to improve yields and specificity.9,10) Of these, dimethyl sulfoxide (DMSO) and formamide are frequently used for the reaction with a GþC-rich DNA to improve specificity, 11,12) but very little is known about the effects of organic solvents on the reverse transcription reaction, although DMSO has been used empirically in the RNA amplification reaction. 4,5) One of the difficult points in this evaluation is that from a practical viewpoint, the efficiency of the reverse transcription reaction is evaluated by the yields and specificity of the subsequent PCR process. Another difficult point is that organic solvents might act on the primer and the RNA as well as on the RT itself.The purpose of this study was to show that organic solvents can improve the efficiency of the RT reaction under certain conditions. We expected that organic solvents ...

Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis

“…The activity of the a-subunit to incorporate dTTP into poly(rA)-p(dT) 15 was performed as described previously (Yasukawa et al 2010b). Briefly, poly(rA)-p(dT) 15 was prepared from p(dT) 15 (Life Technologies Japan Ltd) and poly(rA) (GE Healthcare, Buckinghamshire, UK).…”

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

“…2(B) shows the specific activities of the reverse transcription reaction for WT and all the single variants. The reaction was carried out in 10 mM Tris-HCl buffer (pH 8.3) containing 50 mM KCl, 2 mM dithiothreitol (DTT), 5 mM MgCl 2 , 12.5 μM poly(rA)-p (dT) 15 15 . The RT concentration was determined with Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin (Nacalai Tesque) as standard.…”

“…Regarding this, we have found that glycerol inhibited the inactivation of MMLV RT during incubation at 43°C. 15) Glycerol is an osmolyte that reduces the water activity of the solution. It stabilizes proteins and folds denatured proteins correctly.…”

“…(C) Thermal stability. RT at 50 nM was incubated in 10 mM phosphate buffer (pH 7.6), 2 mM DTT, 0.2% v/v Triton X-100, 10% v/v glycerol, and 28 μM poly(rA)-p(dT) 15 at 48 or 50°C for 10 min. Then a dTTP incorporation reaction was carried out at 37°C.…”

“…Relative activity was defined as the ratio of the initial reaction rate of RT with 10 min incubation at 48 or 50°C to that without the incubation. Note: RT at 50 nM was incubated with 28 μM poly(rA)-p(dT) 15 at 50ºC for 10 min. Then a dTTP incorporation reaction was carried out at 37°C.…”