Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants

Baranauskas, Aurimas; Paliksa, Sigitas; Alzbutas, Gediminas; Vaitkevicius, Mindaugas; Lubienè, Judita; Letukiene, Virginija; Burinskas, Sigitas; Sasnauskas, Giedrius; Skirgaila, Remigijus

doi:10.1093/protein/gzs034

Cited by 68 publications

(59 citation statements)

References 35 publications

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“…To address these problems, a number of derivatives of retroviral RTs have been developed that have increased thermostability and processivity, e.g., Affinityscript (Agilent) (Arezi and Hogrefe 2009), Maxima (ThermoScientific), Rocketscript (Bioneer), Thermoscript (Life Technologies), and Monsterscript (Illumina) or fidelity (AccuScript; Stratagene). An exceptionally improved derivative of M-MLV RT, which contains five mutations, is active at temperatures up to 70°C and has a processivity of 1000-1500 nt on a selected RNA template, but may have somewhat decreased fidelity (error rate reported as <10 −4 ) (Baranauskas et al 2012). Retroviruses are only one of a number of different types of retroelements that are found in nature.…”

Section: Introductionmentioning

confidence: 99%

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Mohr

Ghanem

Smith

et al. 2013

RNA

196

301

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Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

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Section: Introductionmentioning

confidence: 99%

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Mohr

Ghanem

Smith

et al. 2013

RNA

196

301

View full text Add to dashboard Cite

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“…As previously observed (14, 16), the average 3:5 ratio was above 2. This is likely due to inefficiency in the reverse transcription reaction (14, 17, 18). …”

Section: Resultsmentioning

confidence: 99%

Simultaneous Isolation of mRNA and Native Protein From Minute Samples Of Cells

Petersen

Andersen

2014

BioTechniques

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Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization using different protein assays.

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“…7) Its half-life (t 1/2 ) for reverse transcription activity during incubation at 50°C was 492 min, higher than that of WT (40 min). 7) We increased thermostability by introducing positive charges by site-directed mutagenesis at positions that have been implicated in interaction with the templateprimer (T/P), and generated highly stable multiple variants, E286R/E302K/L435R (MM3) and E286R/E302K/ L435R/D524A (MM4). 8) Their T 50 values, of MM3 and MM4, were 54 and 56°C, respectively, higher than that of WT (44°C).…”

mentioning

confidence: 92%

“…We think that the thermostable multiple MMLV RT variants that we 8) and others 6,7) have generated and which we generated in this study are more valuable for various research projects, including transcriptome analysis and clinical diagnosis, than wild-type MMLV RT. However, without a precise comparison of fidelity and processivity, it is difficult to determine which variant is the most suitable.…”

mentioning

confidence: 93%

“…6) Baranauskas et al increased thermostability by random mutagenesis in combination with compartmentalized ribosome display evolution, and generated a highly stable multiple variant, L139P/ D200N/T330P/L603W/E607K. 7) Its half-life (t 1/2 ) for reverse transcription activity during incubation at 50°C was 492 min, higher than that of WT (40 min). 7) We increased thermostability by introducing positive charges by site-directed mutagenesis at positions that have been implicated in interaction with the templateprimer (T/P), and generated highly stable multiple variants, E286R/E302K/L435R (MM3) and E286R/E302K/ L435R/D524A (MM4).…”

mentioning

confidence: 99%

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

Konishi

Yasukawa

2014

Bioscience, Biotechnology, and Biochemistry

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After thermal incubation at 48 °C for 10 min, single variants of Moloney murine leukemia virus reverse transcriptase, V433R and V433K in which a surface hydrophobic residue, Val433, was mutated, retained 55% of initial reverse transcription activity, while the wild-type enzyme retained 17%. After thermal incubation at 50 °C for 10 min, multiple variants D108R/E286R/V433R and D108R/E286R/V433R/D524A, in which Val433→Arg was combined with stabilizing mutations we identified previously, Asp108→Arg and Glu286→Arg, and RNase H activity-eliminating mutation Asp524→Ala, retained 70% of initial activity, exhibiting higher stability than V433R or V433K.

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Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants

Cited by 68 publications

References 35 publications

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Simultaneous Isolation of mRNA and Native Protein From Minute Samples Of Cells

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

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