Improving the affinity and inhibitory characteristics of aptamers is important in the context of their applications in therapy and diagnostics. The use of peptide-aptamer conjugates with an extended aptamer-protein interface is an efficient strategy toward this goal. Here, we report GLE peptide conjugates of the bimodular duplex-quadruplex thrombin aptamers Re31 and NU172. Biophysical studies of the aptamer conjugates revealed that the presence of the tripeptide subunit does not have a significant effect on the thermodynamic stability or structure of the aptamers. In contrast, the affinity and anticoagulant activity of the conjugates were significantly improved. The NU172-GLE conjugate appeared to be the most effective inhibitor of thrombin-induced fibrinogen polymerization. Further clotting studies in human plasma showed that due to the presence of prothrombin, an alternative target for aptamers, the antithrombin activity of aptamers in plasma samples may be significantly underestimated.

Improving the affinity and inhibitory characteristics of aptamers is important in the context of their applications in therapy and diagnostics. The use of peptide-aptamer conjugates with an extended aptamer-protein interface is an efficient strategy toward this goal. Here, we report GLE peptide conjugates of the bimodular duplex-quadruplex thrombin aptamers Re31 and NU172. Biophysical studies of the aptamer conjugates revealed that the presence of the tripeptide subunit does not have a significant effect on the thermodynamic stability or structure of the aptamers. In contrast, the affinity and anticoagulant activity of the conjugates were significantly improved. The NU172-GLE conjugate appeared to be the most effective inhibitor of thrombin-induced fibrinogen polymerization. Further clotting studies in human plasma showed that due to the presence of prothrombin, an alternative target for aptamers, the antithrombin activity of aptamers in plasma samples may be significantly underestimated.

BACKGROUND:Aegilops biuncialisis tetraploid grass species with UbUbMbMb genome constitution, distributed in Mediterranean and the Middle East. It carries many valuable traits such as disease resistance, drought tolerance, high micronutrient content, which are in demand of wheat breeding. Transfer of genetic material from Ae. biuncialis is not easy due to substantial modification of Ub and especially Mb genomes. Cytogenetic markers permitting easy and reliable chromosome identification will be helpful for successful manipulation with alien genetic material and introgression of useful traits into wheat. Development of chromosome nomenclature is complicated by the significant karyotype diversity of Ae. biuncialis. RESULTS:We used various combinations of eleven DNA probes for studying intraspecific karyotype divergence of Ae. biuncialis by FISH; among them pTa-566, pTa713 and pSc119.2 probes proved to be most informative for chromosome identification and analysis of karyotype evolution. FISH discriminated three chromosomal groups designated A, B and C and showed that the Ub genome of Ae. biuncialis is less modified relative to the parental compared to Mb genome. Based on the obtained results we suggested that Ae. biuncialis originated via multiple hybridization events and the Ub and Mb genomes of group A, B, and C were contributed by different forms of Ae. umbellulata and Ae. comosa. The Mb genome of groups A and C probably derived from Ae. comosa subsp. comosa, whereas in the B-group - from subsp. heldreichii. Divergence of chromosomal groups of Ae. biuncialis was also accompanied by structural chromosome rearrangements. Using multiple DNA probes, we showed that reciprocal translocation between chromosomes 1MbL and 7MbL followed by pericentric inversion of modified 1Mb occurred in group A. Intraspecific divergence of Ae. biuncialis was also associated with amplification/ elimination/ redistribution of repetitive DNA families. CONCLUSIONS:Our study revealed complex genome structure of wild tetraploid grass Ae. biuncialis which might have occurred via multiple hybridization events resulting in formation of three distinct chromosomal groups. Their divergence was accompanied by different chromosomal rearrangements which resulted in formation of highly distinct karyotypes. By using FISH markers, we evaluated the relations between chromosomes of the three groups and developed genetic nomenclature of Ae. biuncialis chromosomes.

Modification of synthetic oligonucleotides and DNA is widely used in many applications in the life sciences. However, in most cases, modified DNA cannot be restored to its native state. Here, we report the preparation of a thymidine-inosine dimer building block (TID) for oligonucleotide synthesis. The TID modification supports the functionalization of synthetic oligonucleotides, which can later be removed to restore the DNA strand to its native state. The TID unit allows for a wide spectrum of postsynthetic modifications of oligonucleotides through click chemistry, including conjugation with fluorescent tags and small molecules, preparation of branched oligonucleotide scaffolds, and anchoring to a solid support. Due to the modification of the thymine base, the TID unit reduces the stability of the DNA duplex. We found that the negative effect of internal TID modification on duplex stability does not exceed the same for a single base mismatch. As long as the TID modification is present in the DNA strand, it disrupts its natural functionality. The “caging” effect of TID in the template strand with respect to DNA polymerase was demonstrated in primer extension experiments. Traceless removal of the temporary functional group occurs through oxidative cleavage of the inosine subunit, resulting in the formation of a native DNA strand with the thymine base left at the cleavage site. An anthracene-modified dodecamer oligonucleotide and a branched oligonucleotide scaffold were used to study the cleavage of the reporter group or the oligonucleotide side strand, respectively. It was shown that aqueous tetramethylguanidine efficiently cleaves the oxidized inosine subunit of TID at 37 °C, forming the native DNA strand.

DNA and RNA G-quadruplexes (GQ) are non-canonical G-rich four-stranded structures with proven important roles in major cellular events. DNA GQs are highly polymorphic. Their topology and G-tetrad architecture are strongly influenced by numerous factors. Notable variations in topology and stability between different GQs arise partially from glycosidic bond syn-anti conformational isomerism. The use of unnatural nucleotide analogues with a preferred syn or anti configuration is an efficient approach to control GQ folding, G-tetrad architecture, and stability. Alpha-nucleosides are known to adopt preferably anti-configuration of glycosidic bond. Selective modification of GQs with alpha-2'-deoxyguanosine (αdG) provides a useful tool to manipulate GQ structural parameters and stability. In the GQ core, unnatural αdG residues mimic invariant syn-dG nucleotides and thus drastically reduce the range of possible conformational rearrangements. Understanding the role of αdG modification in controlling GQ architecture and stability requires further biophysical and structural studies of modified GQs. In this paper, we report the design and biophysical characterization of anti-parallel three-layer GQs with αdG-modified G-tetrads of the same polarity. Furthermore, we investigate the therapeutic potential of modified G-rich oligonucleotides in the context of induced formation of αdG-modified anti-parallel GQ. A highly specific termination of primer extension at selected position was induced at a short G3 run in the template strand via guided association with the three segment G-rich modified oligonucleotide.

Objective: Identification of driver mutations in tumors is an extremely important task in oncology for the choice of treatment strategy and assessment of therapy efficacy. In many cases, especially in disease monitoring, there is a need to detect a small number of copies of the mutant allele against the background of excessive content of wild-type DNA. Methods: Genomic DNA was isolated from tumor tissue in paraffine blocks and amplified using polymerase-chain reaction (PCR) with blocking of wild-type DNA amplification via addition of locked-nucleic acid (LNA) oligonucleotides. Fluorescently labelled PCR-product enriched by IDH-mutant alleles was hybridized on a biochip with immobilized oligonucleotide probes which was able to determine 5 mutations in the IDH1 gene and 2 mutations in the IDH2 gene. Results and Discussion: The method was developed and tested on a collection of 26 samples of paraffinized tumor tissue (glioma, glioblastoma, chondrosarcoma). In three cases, R132C, R132L, and R132H mutations in the IDH1 gene were detected in tumor samples with low representation of the mutant allele. The limit of detection of mutant DNA was determined to be 0.1

Objective: The introduction of fluorescently labeled nucleotides in the process of DNA amplification in molecular genetic analysis is a very attractive alternative to post-labeling or the use of labeled primers in the reaction (RCA). Methods: To study the substrate efficiency, the kinetic index (amplification efficiency) was studied, which allowed us to evaluate the inhibitory effect of modified substrates; as well as the yield of the isothermal amplification reaction product and the density of embedding the label into the growing DNA chain. Results and Discussion: Two pairs of Cy5-labeled dU and dC triphosphates with similar electroneutral fluorophore structures, differing in the length of the hydrocarbon linker between the fluorophore and the nitrogenous base were synthesized. A comparative analysis of their substrate behavior in the rolling circle amplification (RCA) reaction using Bst 3.0 DNA polymerase was carried out. It was found that nucleotides with a long linker between the fluorophore and the pyrimidine base are more efficiently incorporated into the growing DNA chain, while the nucleotides with a short linker cause lesser inhibition of RCA. In each of the pairs consisting of dU and dC with similar fluorophores and linkers, the fluorescently labeled uridine derivatives demonstrated a higher incorporation density. It was revealed that, under simultaneous incorporation of the labeled dU and dC, the inhibitory effects do not sum up. This gives grounds for a more careful study of different Cy5-dC variants with a view to increasing the sensitivity of the analysis under simultaneous introduction of the labeled dU and dC. Conclusions: The work presents the results of comparative study of the substrate properties of fluorescently labeled deoxynucleoside triphosphates in RCA. The influence of the chemical nature of the nucleotide (dU or dC) and the structure of the fluorophore on the substrate properties of modified deoxynucleoside triphosphates has been studied. Deoxyuridines with a long linker between the fluorophore and the nitrogenous base were found to be the most effective substrates. In addition, the possibility of simultaneous use of labeled dU and dC in the process of isothermal amplification has been shown, which is potentially capable of increasing the sensitivity of the analysis method.

Objectives. To study the substrate properties of Cy5-labeled deoxynucleoside triphosphates of various natures (dU and dC) in the process of incorporation in the DNA chain during recombinase polymerase amplification (RPA).Methods. The work used the real-time RPA method. The method of horizontal electrophoresis was used to control the quality of the amplification products obtained.Results. The influence of the fluorophore structure and linker lengths on the substrate properties for deoxynucleoside triphosphates Cy5-dUTP and Cy5-dCTP was studied. The following values of the substrate efficiency parameters were determined: amplification efficiency (kinetic indicator), normalized product yield, and embedding coefficient.Conclusions. Modified deoxynucleoside triphosphates (dNTP) with long linkers between the fluorophore and the nitrogenous base, as well as between the quaternary ammonium group and the second heterocycle of the fluorophore, showed greater substrate efficiency than fluorescently labeled dNTP with short linkers. The modified dU in each pair demonstrated greater substrate efficiency compared to the modified dC.

We have previously shown that 5-arylaminouracil derivatives can inhibit HIV-1, herpesviruses, mycobacteria, and other pathogens through various mechanisms. The purpose of this study was to evaluate the potential of 5-arylaminouracils and their derivatives against leukemia, neuroblastoma, and glial brain tumors. 5-Aminouracils with various substituents and their 5'-norcabocyclic and ribo derivatives were screened for cytotoxicity against two neuroblastoma cell lines (SH-SY5Y and IMR-32), K-562 lymphoblastic cells, HL-60 promyeoloblastic cells, and low-passage variants of well-differentiated glioblastoma multiforme (GBM5522 and GBM6138). Cytotoxicity assessment by the standard MTT test showed that most of the compounds lack significant toxicity towards the above cells. However, 5-(4-isopropylphenylamine)uracil and 5‑(4-tert-butylphenylamine)uracil exhibited a dose-dependent toxic effect towards the GBM6138 cell line with half-maximal inhibitory concentrations (IC50) of 9 and 2.3 μM, respectively. Antitumor activity was for the first time demonstrated for compounds of this type and can serve as a starting point for further research.

Introdiction . Systemic chemotherapy (CT) based on oxaliplatin, 5-fluorouracil, capecitabine is the standard of treatment for advanced gastric, colorectal and rectal cancer, which is characterized by frequent development of severe adverse events (AEs). The results of translational studies in the Russian patient population are limited, it is necessary to study pharmacogenetic markers. Aim. To study the frequency of carrying allelic variants of DPYD, GSTP1, MTHFR, XPC, ERCC1, TYMS genes and their association with the development of AEs during palliative treatment with FOLFOX/XELOX. Materials and methods . A total of 166 patients (67 gastric cancer, 99 colorectal cancer) were included in the prospective observational study. All patients underwent pharmacogenetic testing by hybridization analysis on biological microarrays ( DPYD (rs2297595 and rs75017182), MTHFR (rs1801133), XPC (rs2228001), TYMS (rs11280056), ERCC1 (rs3212986)) and PCR ( GSTP1 (rs1695), ERCC1 (rs11615)) before starting CT. The genotype frequency distribution was analyzed between the groups of patients with and without the development of severe AEs. Results . AEs developed in 97.7% of patients, severe AEs accounting for 54.2%. According to the results of univariate analysis, TC genotype of DPYD gene rs2297595 OR = 3.0 (95% CI 1.2–7.3, p = 0.025), GG genotype of GSTP1 gene rs1695 OR = 2.9 (95% CI 1.02–8.6, p = 0.038) were associated with the development of severe neutropenia. In multivariate analysis TT genotype rs2297595 of the DPYD gene remained the only predictor of severe neutropenia (B ± SE = -1.103 ± 0.503; DI [-2.090; -0.116]; p = 0.028). Conclusions . The results of this study allowed us to identify possible markers of toxicity of FOLFOX/XELOX chemotherapy.

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products that are similar in size. Identification of the pathogen was carried out by visual analysis of an electrophoregram. The analytical sensitivity of the developed multiplex RPA was 10(2)-10(3) copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37(rv), and amounted to 100%. The execution time of the analysis is less than an 1 h, including the electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.

Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences.

[This corrects the article DOI: 10.3389/fpls.2022.980764.].

Aegilops comosa Smith in Sibthorp et Smith, 1806 is diploid grass with MM genome constitution occur-ring mainly in Greece. Two morphologically distinct subspecies - Ae. c. comosa Chennaveeraiah, 1960 and Ae. c. heldreichii (Holzmann ex Boissier) Eig, 1929 are discriminated within Ae. comosa, however, genetic and karyotypic bases of their divergence are not fully understood. We used Fluorescence in situ hybridization (FISH) with repetitive DNA probes and electrophoretic analysis of gliadins to character-ize the genome and karyotype of Ae. comosa to assess the level of their genetic diversity and uncover mechanisms leading to radiation of subspecies. We show that two subspecies differ in size and morphol-ogy of chromosomes 3M and 6M, which can be due to reciprocal translocation. Subspecies also differ in the amount and distribution of microsatellite and satellite DNA sequences, the number and position of minor NORs, especially on 3M and 6M, and gliadin spectra mainly in the a-zone. Frequent occurrence of hybrids can be caused by open pollination, which, along with genetic heterogeneity of accessions and, probably, the lack of geographic or genetic barrier between the subspecies, may contribute to extremely broad intraspecific variation of GAAn and gliadin patterns in Ae. comosa, which are usually not observed in endemic plant species.

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase–DNA–dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease in PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.

Aspirin resistance (AR) is a pressing problem in current ischemic stroke care. Although the role of genetic variations is widely considered, the data still remain controversial. Our aim was to investigate the contribution of genetic features to laboratory AR measured through platelet aggregation with arachidonic acid (AA) and adenosine diphosphate (ADP) in ischemic stroke patients. A total of 461 patients were enrolled. Platelet aggregation was measured via light transmission aggregometry. Eighteen single-nucleotide polymorphisms (SNPs) in ITGB3, GPIBA, TBXA2R, ITGA2, PLA2G7, HMOX1, PTGS1, PTGS2, ADRA2A, ABCB1 and PEAR1 genes and the intergenic 9p21.3 region were determined using low-density biochips. We found an association of rs1330344 in the PTGS1 gene with AR and AA-induced platelet aggregation. Rs4311994 in ADRA2A gene also affected AA-induced aggregation, and rs4523 in the TBXA2R gene and rs12041331 in the PEAR1 gene influenced ADP-induced aggregation. Furthermore, the effect of rs1062535 in the ITGA2 gene on NIHSS dynamics during 10 days of treatment was found. The best machine learning (ML) model for AR based on clinical and genetic factors was characterized by AUC = 0.665 and F1-score = 0.628. In conclusion, the association study showed that PTGS1, ADRA2A, TBXA2R and PEAR1 polymorphisms may affect laboratory AR. However, the ML model demonstrated the predominant influence of clinical features.

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

Aegilops crassa Boiss. is polyploid grass species that grows in the eastern part of the Fertile Crescent, Afghanistan, and Middle Asia. It consists of tetraploid (4x) and hexaploid (6x) cytotypes (2n = 4x = 28, D1D (Abdolmalaki et al., 2019) XcrXcr and 2n = 6x = 42, D1D (Abdolmalaki et al., 2019) XcrXcrD2D (Adams and Wendel, 2005), respectively) that are similar morphologically. Although many Aegilops species were used in wheat breeding, the genetic potential of Ae. crassa has not yet been exploited due to its uncertain origin and significant genome modifications. Tetraploid Ae. crassa is thought to be the oldest polyploid Aegilops species, the subgenomes of which still retain some features of its ancient diploid progenitors. The D1 and D2 subgenomes of Ae. crassa were contributed by Aegilopstauschii (2n = 2x = 14, DD), while the Xcr subgenome donor is still unknown. Owing to its ancient origin, Ae. crassa can serve as model for studying genome evolution. Despite this, Ae. crassa is poorly studied genetically and no genome sequences were available for this species. We performed low-coverage genome sequencing of 4x and 6x cytotypes of Ae. crassa, and four Ae. tauschii accessions belonging to different subspecies; diploid wheatgrass Thinopyrum bessarabicum (Jb genome), which is phylogenetically close to D (sub)genome species, was taken as an outgroup. Subsequent data analysis using the pipeline RepeatExplorer2 allowed us to characterize the repeatomes of these species and identify several satellite sequences. Some of these sequences are novel, while others are found to be homologous to already known satellite sequences of Triticeae species. The copy number of satellite repeats in genomes of different species and their subgenome (D1 or Xcr) affinity in Ae. crassa were assessed by means of comparative bioinformatic analysis combined with quantitative PCR (qPCR). Fluorescence in situ hybridization (FISH) was performed to map newly identified satellite repeats on chromosomes of common wheat, Triticum aestivum, 4x and 6x Ae. crassa, Ae. tauschii, and Th. bessarabicum. The new FISH markers can be used in phylogenetic analyses of the Triticeae for chromosome identification and the assessment of their subgenome affinities and for evaluation of genome/chromosome constitution of wide hybrids or polyploid species.