Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

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 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.

Diversity of Self-Assembled RNA Complexes: From Nanoarchitecture to Nanomachines.

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.

Synthesis of the new nucleoside 5'-alpha-iminophosphates using Staudinger reaction.

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.

Structure and hybridization properties of phosphoryl guanidine oligonucleotides under crowding conditions.

Phosphoryl guanidine oligonucleotides (PGOs) are promising uncharged analogs of nucleic acids and are used in a variety of applications. The importance of hydration is frequently ignored during the design of modified nucleic acid probes. Such hydrophobic modifications (phosphoryl guanidine) are expected to have a significant impact on the structure and thermal stability of the affected oligo with complementary nucleic acids. Here we aimed to investigate (by the osmotic stress method) hydration changes upon the formation of a duplex of a PGO with complementary DNA. According to our results, the presence of phosphoryl guanidines in one or both strands of a duplex only minimally affects hydration alterations under crowding conditions. The secondary structure of native and modified duplexes did not change significantly in the presence of ethanol, ethylene glycol, polyethylene glycol 200, or polyethylene glycol 1000. After the addition of a cosolvent, the thermodynamic stability of the PGO complexes changed in the same manner as that seen in a corresponding DNA duplex. The findings reported here and our previous studies form the basis for efficient use of PGOs in basic research and a variety of applications.

DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

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.

Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis.

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

G-quadruplex 2'-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays.

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2'-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2'-F-RNA aptamers. We also demonstrated a possible application of these 2'-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.

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. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.

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.

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.

Complementary-addressed site-directed spin labeling of long natural RNAs.

Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron-electron resonance provided spin-spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.

QCM-based rupture force measurement as a tool to study DNA dehybridization and duplex stability.

The stability of double-stranded DNA (dsDNA) was assessed on the basis of unwinding force measurement. Unwinding force was measured directly with a quartz crystal microbalance (QCM). The amplitude of its surface oscillations was controlled by supplying variable alternate voltage. Under smoothly increasing amplitude of QCM surface oscillations, dsDNA fixed on QCM surface through one of its ends got unwound. This procedure allows reliable measurement of rupture force as small as 5-10 pN. It was demonstrated that oscillations of the surface, with dsDNA bound through one of its ends to this surface, at a frequency of 14 MHz, cause helix unwinding to form two complementary parts due to viscous forces of the liquid medium. Unwinding starts at the upper end. This was proven using oligonucleotide duplexes containing mismatches in different positions. For duplexes containing complementary 20 base pairs, the helix unwinding force is equal to 30-40 pN, which is in agreement with the data obtained by means of atomic-force microscopy (AFM) for the case of unzipping mode. Graphical Abstract Rupture force depending on mismatch position in dsDNA.

Interaction of triarylmethyl radicals with DNA termini revealed by orientation-selective W-band double electron-electron resonance spectroscopy.

Spin labels selectively attached to biomolecules allow high-accuracy nanoscale distance measurements using pulsed electron paramagnetic resonance (EPR), in many cases providing the only access to the structure of complex biosystems. Triarylmethyl (TAM) radicals have recently emerged as a new class of spin labels expanding the applicability of the method to physiological temperatures. Along with other factors, the accuracy of the obtained distances crucially relies on the understanding of interactions between biomolecules and spin labels. In this work, we consider such crucial interactions and their impact on pulsed EPR distance measurements in TAM-labeled DNAs. Using orientation-selective high-frequency (94 GHz) double electron-electron resonance (DEER) we demonstrate strong specific interactions between DNA termini and TAM labels, leading to a significant restriction of their conformational mobility. An understanding of such interactions guides the way to select optimum TAM-labeling strategies, thus refining nanoscale EPR distance measurements in nucleic acids and their complexes under physiological conditions.

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. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

Nanorings from Concatemeric DNA: Chemical Modification Drives Nanostructure Formation.

Self-assembly of DNA concatemers from native duplexes and those containing non-nucleotidic bridges of varying polarity composed of repeating oligo(ethylene glycol) phosphates -O(CH2CH2O)(n)PO2- or α,Ω-alkanediol phosphates -O(CH2)10OPO2(-)- units was compared. The structures obtained were characterised by polyacrylamide gel electrophoresis, enzymatic digestion and AFM. Our results have revealed that chemically-modified duplexes favour self-termination of concatemer growth and yield up to 35% of nanosized DNA rings.

Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy.

Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).

QCM-based measurement of bond rupture forces in DNA double helices for complementarity sensing.

After fixing the DNA molecule in the form of a double helix on the surface of a thickness shear mode resonator (QCM), mechanical oscillations at increasing amplitude cause detorsion of the helix. The force necessary for detorsion can be determined from the voltage applied to the QCM at the rupture moment. The high sensitivity of this method is due to the fact that measurements are carried out in the frequency region around the QCM resonance, where any (even very weak) distortions of the consistent oscillating system cause noticeable distortions of the amplitude-frequency dependence, and these distortions are used to fix the rupture moment. The measured rupture forces were within 30-40 pN, and the sensitivity was 10(8) molecules. It was demonstrated that the proposed procedure allows one to determine the factors that affect the stability of the DNA double helix. This procedure can be the basis for the development of a new method of rapid DNA analysis. Experiments performed with model DNA showed that it is possible to reveal complementarity between two DNA samples.

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties.

In this work, we present new oligonucleotide derivatives containing inter-nucleotide N-benzimidazole, N-benzoxazole, N-benzothiazole, and 1,3-dimethyl-N-benzimidazole (benzoazoles) phosphoramide groups. These modifications were introduced via the Staudinger reaction with appropriate azides during standard automated solid-phase oligonucleotide synthesis. The principal structural difference between the new azido modifiers and those already known is that they are bulk heterocyclic structures, similar to purine nucleoside bases. Modified oligonucleotides with one and two modifications at different positions and multiple modified heteronucleotide sequences were obtained with high yields. The possibility of multiple modifications in the process of automatic DNA synthesis is fundamental and critical for further application of our oligonucleotide derivatives. Initial studies on the properties of new oligonucleotides were carried out. The stability of the oligodeoxyribonucleotide duplex containing phosphoramide groups of N-benzoazoles with complementary DNA or RNA is slightly lower than that of native complexes.