Maxim S. Kupryushkin

A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

Efficient Functionalization of Oligonucleotides by New Achiral Nonnucleosidic Monomers

A novel synthetic strategy has been designed for preparation of achiral nonnucleosidic phosphoramidite monomers for automated solid-phase oligonucleotide synthesis. It is based on O-DMTr-protected 4-(2-hydroxyethyl)-morpholine-2,3-dione as the key compound and a family of building blocks obtained by its ring-opening by primary aliphatic amines. A series of nonnucleosidic phosphoramidites containing various side-chain functionalities was synthesized, and corresponding oligodeoxyribonucleotides incorporating modified units in single or multiple positions along the chain were prepared.

Oligonucleotide Functionalization by a Novel Alkyne-Modified Nonnucleosidic Reagent Obtained by Versatile Building Block Chemistry

A convenient synthetic strategy has been designed to prepare an alkyne-modified synthon for automated DNA synthesis. It is based on the key O-DMTr-protected 4-(2-hydroxyethyl)morpholin-2,3-dione and building blocks obtained by its functionalization by various aliphatic amines. A respective nonnucleosidic phosphoramidite monomer containing a terminal alkyne in the side-chain was synthesized, and corresponding oligothymidylates incorporating the modification in various positions were prepared. The presence of the alkyne group was confirmed by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between the functionalized oligonucleotide and an azide derivative of 7-nitro-2,1,3-benzoxadiazole.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

A new simple and convenient method for preparation of oligonucleotides containing a pyrene or a cholesterol moiety

A new simple and convenient approach to the synthesis of oligonucleotides containing pyrene or cholesterol moieties is described. The approach does not require a special phosphoramidite reagent as it is based on Staudinger reaction between an azidoalkyl derivative of either pyrene or cholesterol and a polymer-supported internucleoside 2-cyanoethyl phosphite, which is formed during phosphoramidite condensation.

A convenient solid phase approach to obtain lipophilic 5′-phosphoramidate derivatives of DNA and RNA oligonucleotides

ABSTRACT This paper explores the potential of a modified phosphotriester approach to the synthesis of 5′-phosphoramidate derivatives of DNA and RNA oligonucleotides. The modification of 5′-deprotected support-bound oligonucleotides is done in two steps: i) conversion of the 5′-OH group of an oligonucleotide into an activated phosphodiester, and ii) treatment of the activated phosphodiester with an aminocompound. The approach is efficient and compatible with conventional solid phase oligonucleotide synthesis. It can be used for the conjugation of therapeutically relevant oligonucleotides with functional moieties or carrier constructions, which are to be removed after endocytosis.

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation

Abstract DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3′ to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.

SDS‐PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs

SDS‐PAGE is considered to be a universal method for size‐based separation and analysis of proteins. In this study, we applied the principle of SDS‐PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, – phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.