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Abstract The dengue protease NS2B/NS3pro has been reported to adopt either an ‘open’ or a ‘closed’ conformation. We have developed a conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution. Experimental values derived from relaxation parameters for the backbone and methyl side chains were compared with the corresponding back-calculated relaxation parameters of different conformational ensembles obtained from free MD simulations. Our results demonstrate a high prevalence for the ‘closed’ conformational ensemble while the ‘open’ conformation is absent, indicating that the latter conformation is most probably due to crystal contacts. Conversely, conformational ensembles in which the positioning of the co-factor NS2B results in a ‘partially’ open conformation, previously described in both MD simulations and X-ray studies, were identified by our conformational filter. Altogether, we believe that our approach allows for unambiguous identification of true conformational ensembles, an essential step for reliable drug discovery.

This review article is focused on the progress made in the synthesis of 5′-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates’ substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research.

Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA.

To date, many derivatives and analogs of nucleic acids (NAs) have been developed. Some of them have found uses in scientific research and biomedical applications. Their effective use is based on the data about their properties. Some of the most important physicochemical properties of oligonucleotides are thermodynamic parameters of the formation of their duplexes with DNA and RNA. These parameters can be calculated only for a few NA derivatives: locked NAs, bridged oligonucleotides, and peptide NAs. Existing predictive approaches are based on an analysis of experimental data and the consequent construction of predictive models. The ongoing pilot studies aimed at devising methods for predicting the properties of NAs by computational modeling techniques are based only on knowledge about the structure of oligonucleotides. In this work, we studied the applicability of the weighted histogram analysis method (WHAM) in combination with umbrella sampling to the calculation of thermodynamic parameters of DNA duplex formation (changes in enthalpy ∆H°, entropy ∆S°, and Gibbs free energy ∆G37° ). A procedure was designed involving WHAM for calculating the hybridization properties of oligodeoxyribonucleotides. Optimal parameters for modeling and calculation of thermodynamic parameters were determined. The feasibility of calculation of ∆H°, ∆S°, and ∆G37° was demonstrated using a representative sample of 21 oligonucleotides 4–16 nucleotides long with a GC content of 14–100 %. Error of the calculation of the thermodynamic parameters was 11.4, 12.9, and 11.8 % for ∆H°, ∆S°, and ∆G37° , respectively, and the melting temperature was predicted with an average error of 5.5 °C. Such high accuracy of computations is comparable with the accuracy of the experimental approach and of other methods for calculating the energy of NA duplex formation. In this paper, the use of WHAM for computation of the energy of DNA duplex formation was systematically investigated for the first time. Our results show that a reliable calculation of the hybridization parameters of new NA derivatives is possible, including derivatives not yet synthesized. This work opens up new horizons for a rational design of constructs based on NAs for solving problems in biomedicine and biotechnology.

New tool development for various nucleic acid applications is an essential task in RNA nanotechnology. Here, we determined the ability of self-limited complex formation by a pair of oligoribonucleotides carrying two pairwise complementary blocks connected by a linker of different lengths in each chain. The complexes were analyzed using UV melting, gel shift assay analysis, and molecular dynamics (MD) simulations. We have demonstrated the spontaneous formation of various self-limited and concatemer complexes. The linear concatemer complex is formed by a pair of oligomers without a linker in at least one of them. Longer linkers resulted in the formation of circular complexes. The self-limited complexes formation was confirmed using the toehold strand displacement. The MD simulations indicate the reliability of the complexes’ structure and demonstrate their dynamics, which increase with the rise of complex size. The linearization of 2D circular complexes into 1D structures and a reverse cyclization process were demonstrated using a toehold-mediated approach. The approach proposed here for the construction and directed modification of the molecularity and shape of complexes will be a valuable tool in RNA nanotechnology, especially for the rational design of therapeutic nucleic acids with high target specificity and the programmable response of the immune system of organisms.

This review article is focused on the progress made in the synthesis of 5′-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates' substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research. [ABSTRACT FROM AUTHOR]

Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes. To explore the reliability of PELDOR and MD simulations, we site-specifically attached nitroxide spin labels to oligonucleotides, which form self-assembled complexes between NA chains and exhibit significant conformational flexibility. The DNA complexes assembled from a pair of oligonucleotides with different linker sizes showed excellent agreement between the distance distributions obtained from PELDOR and calculated from MD simulations, both for the mean inter-spin distance and the distance distribution width. These results prove that PELDOR with MD simulations has significant potential for studying the structure and dynamics of conformational flexible complexes of NA. [ABSTRACT FROM AUTHOR]

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer’s 3′-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template’s background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

This article presents new data on nucleotide incision repair (NIR) activity of apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae, which is known as a key player of the base excision DNA repair (BER) pathway, see “Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV” [1], “Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae” [2] and “Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases” [3]. The characterization of NIR activity of wild type Apn1 and mutant form Ape1 H83A were made by denaturing PAGE analysis, and MD simulations of Apn1 complexed with DNA containing 5,6-dihydro-2′-deoxyuridine (DHU) and 2-aminopurine (2-aPu) residues. This data article is associated to the manuscript titled “Apurinic/apyrimidinic endonuclease Apn1 from Saccharomyces cerevisiae is recruited to the nucleotide incision repair pathway: kinetic and structural features” [4].Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

New tool development for various nucleic acid applications is an essential task in RNA nanotechnology. Here, we determined the ability of self-limited complex formation by a pair of oligoribonucleotides carrying two pairwise complementary blocks connected by a linker of different lengths in each chain. The complexes were analyzed using UV melting, gel shift assay analysis, and molecular dynamics (MD) simulations. We have demonstrated the spontaneous formation of various self-limited and concatemer complexes. The linear concatemer complex is formed by a pair of oligomers without a linker in at least one of them. Longer linkers resulted in the formation of circular complexes. The self-limited complexes formation was confirmed using the toehold strand displacement. The MD simulations indicate the reliability of the complexes' structure and demonstrate their dynamics, which increase with the rise of complex size. The linearization of 2D circular complexes into 1D structures and a reverse cyclization process were demonstrated using a toehold-mediated approach. The approach proposed here for the construction and directed modification of the molecularity and shape of complexes will be a valuable tool in RNA nanotechnology, especially for the rational design of therapeutic nucleic acids with high target specificity and the programmable response of the immune system of organisms. [ABSTRACT FROM AUTHOR]

This article presents new data on the properties of the diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotides d(TpCp*A) [1,2]. The data include information on isolation, identification, treatment with snake venom phosphodiesterase and structural analysis by 1D and 2D NMR spectroscopy and restrained molecular dynamics analysis. The data can be used for preparation, analysis, application of phosphoryl guanidine oligonucleotide and for development of new nucleic acids derivatives. This data article is associated with the manuscript titled “Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: isolation and properties” [1]. Keywords: Phosphoryl guanidine oligonucleotide, RP-HPLC, Circular dichroism, NMR, Molecular dynamics, Diastereomer assignment phosphodiesterase digestion assay, Spatial structure

The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19F ENDOR experiments on reporting intermolecular distances in trityl- and 19F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end. [ABSTRACT FROM AUTHOR]

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

We demonstrated that it is possible to break apart double-stranded DNA using a thickness shear mode resonator. This procedure measures the force of bond rupture. The measured rupture forces were in the range of 10–40 pN. The procedure was tested experimentally with model complementary and non-complementary oligonucleotides. In the case of the DNA mismatch we observed a signal at a driving voltage of 0.8 V (corresponding to a shear force of 11 pN), while in the case of complementarity the signal at the voltage of 3 V (36 pN) appeared. This procedure may be the ground for the development of new types of biosensors measuring the force of affine interactions (probe/biomolecular target) on the surface of the sensor element.