Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli.

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

Analysis of RNA by Primer Extension.

For mapping the 5' termini of mRNA molecules, primer extension is the method of choice. A purified oligonucleotide is end-labeled using polynucleotide kinase. The probe and a population of mRNA are allowed to hybridize, and the primers and template are used to carry out reverse transcription using an enzyme cloned from the Moloney murine leukemia virus. The primer extension products are separated on a denaturing polyacrylamide gel and analyzed by radiography.

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/µL of input viral genomic RNA.

The attachment of a DNA-binding Sso7d-like protein improves processivity and resistance to inhibitors of M-MuLV reverse transcriptase.

Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives.

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.

Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases.

We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus 1 (HIV-1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(DEA tC)TP as substrates. Michaelis-Menten kinetics for the insertion of these analogues show Vmax /KM from 0.0-5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV-1-RT, including RNA-dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(DEA tC)TP are compatible with a complete reverse transcription cycle in vitro.

Integrating molecular dynamics simulation and molecular mechanics/generalized Born surface area calculation into pharmacophore modeling: a case study on the proviral integration site for Moloney murine leukemia virus (Pim)-1 kinase inhibitors.

Communicated by Ramaswamy H. Sarma.

Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other.

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.

Dual coding potential of a 2',5'-branched ribonucleotide in DNA.

Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2',5'-branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific. DNA synthesis from the 2'-arm is more than 20,000-fold decreased, whereas from the 3'-arm only 15-fold. Our sequence analysis of full-length nucleic acid generated by Taq DNA polymerase, Moloney murine leukemia virus reverse transcriptase, and T7 RNA polymerase from the 2'-arm of bDNA shows that the branched guanine has a dual coding potential and can base-pair with cytosine and guanine. We find that branchpoint templating is influenced by the type of the surrounding nucleic acid and is probably modulated by polymerase and RNase H active sites. We show that the branchpoint bypass by the polymerases from the 3'-arm of bDNA is predominantly error-free, indicating that bDNA is not as highly mutagenic as 2',5'-branched RNA.

Capping-RACE: a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Rapid amplification of cDNA ends (RACE) is a prevalent technique used to obtain the 5' ends of transcripts. Several different 5' RACE methods have been developed, and one particularly simple and efficient approach called CapFinder relies on the 5' cap-dependent template-switching that occurs in eukaryotes. However, most prokaryotic transcripts lack a 5' cap structure. Here, we report a procedure to capture primary transcripts based on capping the 5' triphosphorylated RNA in prokaryotes. Primary transcripts were first treated with vaccinia capping enzyme to add a 5' cap structure. First-strand cDNA was then synthesized using Moloney murine leukaemia virus reverse transcriptase. Finally, a template-switching oligonucleotide with a tail containing three ribonucleic acid guanines was hybridized to the cDNA 3' poly(C) and further used as template for reverse transcriptase. It is oligonucleotide sequence independent and is more sensitive compared to RLM-RACE. This approach specifically identified the transcription start sites of ompA, sodB and shiA in Escherichia coli and of ompA, rne and rppH in Brucella melitensis. Furthermore, we also successfully identified the transcription start sites of small noncoding genes ryhB and micC in E. coli and bsnc135 and bsnc149 in B. melitensis. Our findings suggest that Capping-RACE is a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase.

In this study, we investigated CIS reaction (clamping-mediated incorporation of single-stranded DNA with concomitant DNA syntheses) of Moloney murine leukaemia virus reverse transcriptase (MMLV-RT), and established a set of conditions with which single-stranded DNA is ligated to a G-tailed model substrate DNA at efficiencies close to 100%. Prior to the CIS reaction, a target blunt-end DNA was 3' G-tailed by MMLV-RT in the presence of a tailing enhancer, deoxycytidine. In the CIS reaction, the G-tail reacted with a single-stranded DNA carrying a stretch of Cs on its 3' end (termed as GAO for guide adaptor oligonucleotide), and MMLV-RT performed DNA polymerization, starting from the 3' overhang, using the GAO as a template. We could append a given nucleotide sequence of as long as 72 nucleotides, which would be sufficient for various NGS-sequencing platforms. The high efficiency and the unique features of this MMLV-RT activity that enables the labelling of each DNA molecule with a unique degenerate sequence as a molecular identifier has many potential uses in biotechnology.

Decreased Km to dNTPs is an essential M-MuLV reverse transcriptase adoption required to perform efficient cDNA synthesis in One-Step RT-PCR assay.

Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase. The most frequent mutations found in a selected library were analyzed. Aside from the mutations, which switch off RNase H activity of RT and are beneficial for the full-length cDNA synthesis, we have identified several mutations in the active center of the enzyme (Q221R and V223A/M), which result in 4-5-fold decrease of Km for dNTPs (<0.2 mM). The selected mutations are in surprising agreement with the natural evolution process because they transformed the active center from the oncoretroviral M-MuLV RT-type to the lenitiviral enzyme-type. We believe that this was the major and essential phenotypic adjustment required to perform fast and efficient cDNA synthesis in PCR buffer at 0.2-mM concentration of each dNTP.

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition.

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Further increase in thermostability of Moloney murine leukemia virus reverse transcriptase by mutational combination.

We previously generated a highly thermostable triple variant of Moloney murine leukemia virus reverse transcriptase, MM3 (E286R/E302K/L435R), by introducing positive charges by site-directed mutagenesis at positions that have been implicated in the interaction with template-primer (Yasukawa et al., (2010) J. Biotechnol., 150, 299-306). In this study, we attempted to further increase the thermostability of MM3. Twenty-nine mutations were newly designed, focusing on the number of surface charge, stabilization of hydrophobic core, and introduction of salt bridge. The corresponding 29 single variants were produced in Escherichia coli and characterized for activity and stability. Six mutations (A32V, L41D, L72R, I212R, L272E and W388R) were selected as the candidates for further stabilize MM3. Fifteen multiple variants were designed by combining two or more of the six mutations with the MM3 mutations, produced and characterized. The sextuple variant MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R) exhibited higher thermostability than MM3.

Next-generation sequencing-based analysis of reverse transcriptase fidelity.

In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 × 10-4 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10-4 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation.

Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase.

In a previous study, we showed that MMLV-RT has a strong terminal transferase activity, and that the C-, G-, and T-tailing activities are enhanced by dGMP, dCMP, and dAMP, respectively. In this study, to achieve faster reaction and higher tailing efficiency, we screened other compounds for the ability to enhance the tailing activities of MMLV-RT, and determined the corresponding optimal concentrations. The C-, G-, and T-tailing activities were enhanced by guanine, cytosine, and adenine, respectively, and by derivatives thereof, suggesting a transient Watson-Click base pairing between an enhancer molecule and the nucleotide to be incorporated. In the presence of some additives (GMP and GDP for C-tailing and CMP for G-tailing), the tail length increased continuously, resulting in tail lengths of 7 to 15 (GMP and GDP) or 13 to 22 (CMP) nucleotides. Among the compounds that do not induce continuous addition, adenosine, deoxycytidine, and deoxyguanosine mostly enhanced T-, G-, and C-tailings, respectively. The enhancing chemicals described here will improve the feasibility of N-tailing by MMLV-RT in various biotechnological applications.