Structural stability of DNA in nonaqueous solvents

Bonner, Gary; Klibanov, Alexander M.

doi:10.1002/(sici)1097-0290(20000505)68:3<339::aid-bit12>3.0.co;2-o

Cited by 111 publications

(95 citation statements)

References 21 publications

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“…1e compares the magic angle fluorescence kinetic traces of two 2-AP-labelled nucleotides, in HEPES and in a (70/30) glycerol/water mixture. We observe that the fastest, static quenching components are strongly reduced in amplitude, indicating that enhanced hydrogen-bonding interactions with the solvent significantly perturb stacking interactions of 2-AP with its nearest neighbours, as suggested for similar glycerol-induced destabilisation effects observed for single stranded DNA [7]. In addition, the residual quenching dynamics is no longer site-specific in the presence of glycerol, indicating that the interactions with the solvent dominate.…”

Section: -P2supporting

confidence: 60%

Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

Touceda

Gelot

Crégut

et al. 2013

EPJ Web of Conferences

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Section: -P2supporting

confidence: 60%

Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

Touceda

Gelot

Crégut

et al. 2013

EPJ Web of Conferences

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“…In relation to this, Bonner and Klibanov found that DNA is completely denatured even at 25 C in 99% DMSO or formamide. 17) McConaughy et al found that the T m of a high molecular DNA-DNA duplex as assessed by the absorbance at 260 nm (A 260 ) was 90 C in the absence of an organic solvent, decreased linearly with increasing formamide concentrations, and reached 45 C in the presence of 65% formamide. 18) Several studies have reported on the effects of DMSO and formamide on PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Rariy and Klibanov found that unlike DMSO and formamide, glycerol stabilizes proteins and folds denatured protein correctly. 25) Bonner and Klibanov found that DNA maintains a duplex structure at 25 C in 99% glycerol, 17) indicating that the effects of glycerol on protein and DNA are much different from those of DMSO and formamide.…”

Section: Discussionmentioning

confidence: 99%

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

Yasukawa

Konishi

Inouye

2010

Bioscience, Biotechnology, and Biochemistry

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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 ...

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“…Nucleic acids allow the use of a wide variety of buffers and organic solvents, which can be interesting for monitoring organic compounds. Indeed, nucleic acids remain stable in hydrogen bond forming solvents such as ethylene glycol, methanol, formamide, dimethyl sulfoxide or acetamide (Bonner & Klibanov 2000). As opposed to their protein counterparts, aptamers can be selected under nonphysiological conditions or real matrix conditions, which is particularly useful for biosensing clinical and food samples.…”

Section: Advantages and Limitations Of Phages And Aptamersmentioning

confidence: 99%

The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and Food Diagnostics

Mehta¹,

Dorst²,

Devriese³

et al. 2011

Biomedical Engineering, Trends, Research and Technologies

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Structural stability of DNA in nonaqueous solvents

Cited by 111 publications

References 21 publications

Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

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

The Use of Phages and Aptamers as Alternatives to Antibodies in Medical and Food Diagnostics

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