Effect of the Phosphoryl Guanidine Modification in Chimeric DNA-RNA crRNAs on the Activity of the CRISPR-Cas9 System .

Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects.

Influence of Combinations of Lipophilic and Phosphate Backbone Modifications on Cellular Uptake of Modified Oligonucleotides.

Numerous types of oligonucleotide modifications have been developed since automated synthesis of DNA/RNA became a common instrument in the creation of synthetic oligonucleotides. Despite the growing number of types of oligonucleotide modifications under development, only a few of them and, moreover, their combinations have been studied widely enough in terms of their influence on the properties of corresponding NA constructions. In the present study, a number of oligonucleotides with combinations of 3'-end lipophilic (a single cholesteryl or a pair of dodecyl residues) and phosphate backbone modifications were synthesized.

Fork- and Comb-like Lipophilic Structures: Different Chemical Approaches to the Synthesis of Oligonucleotides with Multiple Dodecyl Residues.

Lipophilic oligonucleotide conjugates represent a powerful tool for nucleic acid cellular delivery, and many methods for their synthesis have been developed over the past few decades. In the present study, a number of chemical approaches for the synthesis of different fork- and comb-like dodecyl-containing oligonucleotide structures were performed, including use of non-nucleotide units and different types of phosphate modifications such as alkyl phosphoramidate, phosphoryl guanidine, and triazinyl phosphoramidate. The influence of the number of introduced lipophilic residues, their mutual arrangement, and the type of formed modification backbone on cell penetration was evaluated.

Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin.

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.

The Lipophilic Purine Nucleoside-Tdp1 Inhibitor-Enhances DNA Damage Induced by Topotecan In Vitro and Potentiates the Antitumor Effect of Topotecan In Vivo.

The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells.

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone.

An Influence of Modification with Phosphoryl Guanidine Combined with a 2'-O-Methyl or 2'-Fluoro Group on the Small-Interfering-RNA Effect.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex.

Amphiphilic "Like-a-Brush" Oligonucleotide Conjugates with Three Dodecyl Chains: Self-Assembly Features of Novel Scaffold Compounds for Nucleic Acids Delivery.

The conjugation of lipophilic groups to oligonucleotides is a promising approach for improving nucleic acid-based therapeutics' intracellular delivery. Lipid oligonucleotide conjugates can self-aggregate in aqueous solution, which gains much attention due to the formation of micellar particles suitable for cell endocytosis. Here, we describe self-association features of novel "like-a-brush" oligonucleotide conjugates bearing three dodecyl chains.

Transport Oligonucleotides-A Novel System for Intracellular Delivery of Antisense Therapeutics.

Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized.

Data on multimerization efficiency for short linear DNA templates and phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase.

This article reports experimental data related to the research article entitled "Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase" (R.R. Garafutdinov, A.

Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase.

Over the last two decades, isothermal amplification of nucleic acids has gained more attention due to a number of advantages over the widely used polymerase chain reaction. For isothermal amplification, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most commonly used. Unfortunately, Bst exo- causes nonspecific DNA amplification (so-called multimerization) under isothermal conditions that results in undesirable products (multimers) consisting of tandem nucleotide repeats.

Data for isolation and properties analysis of diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide.

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.

Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties.

Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers ('fast' and 'slow') of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC.

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

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.

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

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.

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.

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).

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.

New oligonucleotide derivatives as unreactive substrate analogues and potential inhibitors of human apurinic/apyrimidinic endonuclease APE1.

Human apurinic/apyrimidinic endonuclease APE1 is one of the key enzymes of the base excision DNA repair system. The main biological function of APE1 is the hydrolysis of the phosphodiester bond on the 5'-side of an apurinic/apyrimidinic site (AP-site) to give the 5'-phosphate and 3'-hydroxyl group. It has long been known that AP-sites have mutagenic and cytotoxic effects and their accumulation in DNA is a potential hazard to the cell lifecycle.

Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay.

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.