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

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. Many approaches focus on modifying components of the system, namely, Cas9 and guide RNAs, to enhance specificity. However, a common challenge is that methods aiming to increase specificity often result in a significant reduction in the editing efficiency. Here, we introduce a novel approach to modifying crRNA to balance CRISPR-Cas9 specificity and efficiency. Our approach involves incorporating nucleoside modifications, such as replacing ribo- to deoxyribonucleosides and backbone modifications, using phosphoryl guanidine groups, specifically 1,3-dimethylimidazolidin-2-ylidene phosphoramidate. In this case, within the first 10 nucleotides from the 5' crRNA end, phosphodiester bonds are substituted with phosphoryl guanidine groups. We demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro.

Fixation and Visualization of Full Protein Corona on Lipid Surface of Composite Nanoconstruction.

Spontaneous sorption of proteins on the nanoparticles' surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins-the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

Chemical Modifications Influence the Number of siRNA Molecules Adsorbed on Gold Nanoparticles and the Efficiency of Downregulation of a Target Protein.

Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2'-OMe and 2'-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition.

A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization.

There is an urgent need to develop systems for nucleic acid delivery, especially for the creation of effective therapeutics against various diseases. We have previously shown the feasibility of efficient delivery of small interfering RNA by means of gold nanoparticle-based multilayer nanoconstructs (MLNCs) for suppressing reporter protein synthesis. The present work is aimed at improving the quality of preparations of desired MLNCs, and for this purpose, optimal conditions for their multistep fabrication were found. All steps of this process and MLNC purification were verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification quality were identified. These data made it possible to optimize the fabrication of target MLNCs bearing small interfering RNA and to substantially improve end product quality via an increase in its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the proposed approaches and methods will be useful for researchers working with lipid nanoconstructs.

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. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

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. The self-assembly of the conjugates into 30-170 nm micellar particles with a high tendency to aggregate was shown using dynamic light scattering (DLS), atomic force (AFM), and transmission electron (TEM) microscopies. Fluorescently labeled conjugates demonstrated significant quenching of fluorescence intensity (up to 90%) under micelle formation conditions. The conjugates possess increased binding affinity to serum albumin as compared with free oligonucleotides. The dodecyl oligonucleotide conjugate and its duplex efficiently internalized and accumulated into HepG2 cells' cytoplasm without any transfection agent. It was shown that the addition of serum albumin or fetal bovine serum to the medium decreased oligonucleotide uptake efficacy (by 22.5-36%) but did not completely inhibit cell penetration. The obtained results allow considering dodecyl-containing oligonucleotides as scaffold compounds for engineering nucleic acid delivery vehicles.

Interaction of Hydrophobic Tungsten Cluster Complexes with a Phospholipid Bilayer.

Nanoconstructions composed of lipid vesicles and inorganic units (nanoparticles, metal complexes) arouse much interest across materials science and nanotechnology as hybrid materials combining useful functionalities from both parts. Ideally, these units are to be embedded into the bilayer to keep the biophysical performance of lipid vesicles having inorganic moieties screened from the environment. This can be achieved by doping a lipid bilayer with cluster complexes of transition metals. In this work, we report the preparation of nanoparticles from trinuclear W3S4 cluster complexes and egg phosphatidylcholine. A systematic study of their properties was performed by the differential scanning calorimetry, NMR spectroscopy, dynamic light scattering, and transmission electron microscopy. Phospholipids and clusters have been found to spontaneously self-assemble into novel cluster-lipid hybrid materials. The behavior of clusters in the hydrophobic lipid environment is determined by the structure of the ligands and cluster-to-lipid ratio. Intact cluster complexes bearing compact hydrophobic ligands are embedded into the hydrophobic midplane of a lipid bilayer, whereas cluster complexes bearing larger ligands drive the aggregation of lipids and cluster complexes. Considering these differences, it could be possible to obtain different self-assembled associates such as cluster-doped liposomes or lipid-covered crystals. These cluster-lipid hybrids can be a platform for the design of new materials for nanotechnology.

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. This data article is associated with the manuscript titled "Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: isolation and properties" [1].

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. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both 'fast' and 'slow' diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Overhauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the 'fast' isomer is the Sp diastereomer, and the 'slow' isomer is the Rp diastereomer.

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. It can be used for the conjugation of therapeutically relevant oligonucleotides with functional moieties or carrier constructions, which are to be removed after endocytosis.