The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents

Nakano, Shu-ichi; Sugimoto, Naoki

doi:10.1007/s12551-015-0188-0

Cited by 77 publications

(80 citation statements)

References 125 publications

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“…According to the literature, PEG2000 and similar molecules do not produce significant excluded volume effects, but they do show different degrees of hydrogen bonding, polarity, and hydrophobicity. Therefore, the addition of PEG2000 to water could create co‐solvent–co‐solvent and water–co‐solvent interactions and interrupt water–water interactions that stabilise nucleic acid complexes . However, upon comparing the ON5+ON6 and ON7+ON8 systems, it was found that the system with the Cy3/Cy5 pair was more stable upon adding polylysine and PEG.…”

Section: Resultssupporting

confidence: 89%

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 μm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.

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Section: Resultssupporting

confidence: 89%

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

et al. 2018

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“…This result indicates that many kinds of organic additives are useful for enhancing the turnover activity of the hammerhead ribozyme. It has been reported that the stability of base pairing and tertiary folding is reduced in the presence of organic solvents, particularly amide compounds that are known to substantially destabilize nucleic acid structures [31,44]. However, this destabilization effect seemed not to cause a disruption of interactions necessary for the formation of the active conformation, such as a network of hydrogen bonds [48], in the presence of 10 mM MgCl 2 .…”

Section: Effects On the Hammerhead Ribozymementioning

confidence: 99%

“…These organic additives may alter the physical properties of solution, interact with nucleic acids, and introduce areas of excluded volume [20,31]. Most mixed solutions have lower dielectric constant values than pure water, and the additives, such as large PEGs, MME, DME, EtOH PrOH, and DOX, significantly lowers the dielectric constant of solution.…”

Section: Classification Of the Mixed Solutions Based On The Effects Omentioning

confidence: 99%

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

2017

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Catalytic nucleic acids are regarded as potential therapeutic agents and biosensors. The catalytic activities of nucleic acid enzymes are usually investigated in dilute aqueous solutions, although the physical properties of the reaction environment inside living cells and that in the area proximal to the surface of biosensors in which they operate are quite different from those of pure water. The effect of the molecular environment is also an important focus of research aimed at improving and expanding nucleic acid function by addition of organic solvents to aqueous solutions. In this study, the catalytic activities of RNA and DNA enzymes (hammerhead ribozyme, 17E DNAzyme, R3C ribozyme, and 9DB1 DNAzyme) were investigated using 21 different mixed aqueous solutions comprising organic compounds. Kinetic measurements indicated that these enzymes can display enhanced catalytic activity in mixed solutions with respect to the solution containing no organic additives. Correlation analyses revealed that the turnover rate of the reaction catalyzed by hammerhead ribozyme increased in a medium with a lower dielectric constant than water, and the turnover rate of the reaction catalyzed by 17E DNAzyme increased in conditions that increased the strength of DNA interactions. On the other hand, R3C ribozyme and 9DB1 DNAzyme displayed no significant turnover activity, but their single-turnover rates increased in many mixed solutions. Our data provide insight into the activity of catalytic nucleic acids under various conditions that are applicable to the medical and technology fields, such as in living cells and in biosensors.

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“…With the use of crowder molecules such as poly(ethylene glycol)s (PEGs) to mimic intracellular crowding conditions, it has been clarified that DNA duplex thermal stabilities decrease due to a decrease in the water activity [ 15 , 16 , 17 ]. Molecular crowding also decreases the dielectric constant of the solution, which can increase the ribozyme activities [ 18 , 19 ]. In polymerase reactions, we found that the efficiency and preference of RNA primer extension along the RNA or DNA template by RNA polymerases were drastically affected by molecular crowding [ 20 ]: T7 RNA polymerase preferentially incorporated dNTPs over NTPs along the RNA template under specific crowding conditions [ 20 ].…”

Section: Introductionmentioning

confidence: 99%

Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

2020

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Unnatural nucleic acids are promising materials to expand genetic information beyond the natural bases. During replication, substrate nucleotide incorporation should be strictly controlled for optimal base pairing with template strand bases. Base-pairing interactions occur via hydrogen bonding and base stacking, which could be perturbed by the chemical environment. Although unnatural nucleobases and sugar moieties have undergone extensive structural improvement for intended polymerization, the chemical environmental effect on the reaction is less understood. In this study, we investigated how molecular crowding could affect native DNA polymerization along various templates comprising unnatural nucleobases and sugars. Under non-crowding conditions, the preferred incorporation efficiency of pyrimidine deoxynucleotide triphosphates (dNTPs) by the Klenow fragment (KF) was generally high with low fidelity, whereas that of purine dNTPs was the opposite. However, under crowding conditions, the efficiency remained almost unchanged with varying preferences in each case. These results suggest that hydrogen bonding and base-stacking interactions could be perturbed by crowding conditions in the bulk solution and polymerase active center during transient base pairing before polymerization. This study highlights that unintended dNTP incorporation against unnatural nucleosides could be differentiated in cases of intracellular reactions.

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The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents

Cited by 77 publications

References 125 publications

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

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