Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Complete genome sequence of new Microbacterium sp. strain ET2, isolated from roots of leafless orchid.

Whole-genome sequence of ET2 strain, isolated from the roots of leafless orchid, constitutes a single circular chromosome of 3,604,840 bp (69.44% G + C content). BLAST+-based average nucleotide identity (ANIb) and digital DNA-DNA hybridization values indicate that ET2 may be a novel Microbacterium species. Genes putatively involved in plant-microbial interactions were predicted.

Conserved G-Quadruplex-Forming Sequences in Mammalian TERT Promoters and Their Effect on Mutation Frequency.

Somatic mutations in the promoter region of the human telomerase reverse transcriptase (hTERT) gene have been identified in many types of cancer. The hTERT promoter is known to be enriched with sequences that enable the formation of G-quadruplex (G4) structures, whose presence is associated with elevated mutagenicity and genome instability. Here, we used a bioinformatics tool (QGRS mapper) to search for G4-forming sequences (G4 motifs) in the 1000 bp TERT promoter regions of 141 mammalian species belonging to 20 orders, 5 of which, including primates and predators, contain more than 10 species. Groups of conserved G4 motifs and single-nucleotide variants within these groups were discovered using a block alignment approach (based on the Nucleotide PanGenome explorer). It has been shown that: (i) G4 motifs are predominantly located in the region proximal to the transcription start site (up to 400 bp) and are over-represented on the non-coding strand of the TERT promoters, (ii) 11 to 22% of the G4 motifs found are evolutionarily conserved across the related organisms, and (iii) a statistically significant higher frequency of nucleotide substitutions in the conserved G4 motifs compared to the surrounding regions was confirmed only for the order Primates. These data support the assumption that G4s can interfere with the DNA repair process and affect the evolutionary adaptation of organisms and species.

Simplex Droplet Digital PCR Assays for the Detection of TERT Promoter Mutations in Urine Samples for the Non-invasive Diagnosis of Urothelial Cancer.

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter region are highly frequent in urothelial cancer (UC), and their detection in urine (cell-free DNA from the urine supernatant or DNA from exfoliated cells in the urine pellet) has demonstrated promising evidence as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumour-derived mutations in urine requires highly sensitive methods, capable of measuring low-allelic fraction mutations. We developed sensitive droplet digital PCR (ddPCR) assays for detecting urinary TERT promoter mutations (uTERTpm), targeting the two most common mutations (C228T and C250T), as well as the rare A161C, C228A, and CC242-243TT mutations. Here, we described the step-by-step protocol uTERTpm mutation screening using simplex ddPCR assays and give some recommendations for isolation of DNA from urine samples. We also provide limits of detection for the two most frequent mutations and discuss advantages of the method for clinical implementation of the assays for the detection and monitoring of UC.

The TERT Promoter: A Key Player in the Fight for Cancer Cell Immortality.

The review describes the role of telomeres and telomerase in tumor progression, as well as various mechanisms of the activation of telomerase reverse transcriptase (TERT) expression in CNS tumors and other cancers. The main mechanism of TERT activation involves acquisition of somatic mutations by the TERT gene promoter (TERTp). The article presents information on the TERTp structure and transcription factors directly interacting with TERTp and regulating its transcription. The prospects of using the mutational status of TERTp as a prognostic marker of CNS malignancies and other tumors with a common profile of TERTp mutations are discussed.

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Draft Genome Sequence of Thermomicrobium sp. Strain 4228-Ro, a Thermophilic Bacterium Isolated from a Kamchatka Hot Spring.

The genome of Thermomicrobium sp. strain 4228-Ro, an aerobic thermophilic bacterium isolated from a Kamchatka hot spring, was sequenced and analyzed. The genome assembly comprises 13 contigs with a total length of 3,068,448 bp. Genome analysis revealed the pathway of aerobic utilization of sugars, which was corroborated by growth experiments.

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure.

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.

TERT Promoter Mutations as Simple and Non-Invasive Urinary Biomarkers for the Detection of Urothelial Bladder Cancer in a High-Risk Region.

Bladder cancer (BC) is the 10th most common cancer in the world. While there are FDA-approved urinary assays to detect BC, none have demonstrated sufficient sensitivity and specificity to be integrated into clinical practice. Telomerase Reverse Transcriptase (TERT) gene mutations have been identified as the most common BC mutations that could potentially be used as non-invasive urinary biomarkers to detect BC. This study aims to evaluate the validity of these tests to detect BC in the Kerman province of Iran, where BC is the most common cancer in men. Urine samples of 31 patients with primary (n = 11) or recurrent (n = 20) bladder tumor and 50 controls were prospectively collected. Total urinary DNA was screened for the TERT promoter mutations (uTERTpm) by Droplet Digital PCR (ddPCR) assays. The performance characteristics of uTERTpm and the influence by disease stage and grade were compared to urine cytology results. The uTERTpm was 100% sensitive and 88% specific to detect primary BC, while it was 50% sensitive and 88% specific in detecting recurrent BC. The overall sensitivity and specificity of uTERTpm to detect bladder cancer were 67.7% and 88.0%, respectively, which were consistent across different tumor stages and grades. The most frequent uTERTpm mutations among BC cases were C228T (18/31), C250T (4/31), and C158A (1/31) with mutant allelic frequency (MAF) ranging from 0.2% to 63.3%. Urine cytology demonstrated a similar sensitivity (67.7%), but lower specificity (62.0%) than uTERTpm in detecting BC. Combined uTERTpm and urine cytology increased the sensitivity to 83.8%, but decreased the specificity to 52.0%. Our study demonstrated promising diagnostic accuracy for the uTERTpm as a non-invasive urinary biomarker to detect, in particular, primary BC in this population.

Model of the SARS-CoV-2 Virus for Development of a DNA-Modified, Surface-Enhanced Raman Spectroscopy Sensor with a Novel Hybrid Plasmonic Platform in Sandwich Mode.

The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has posed a great challenge for the development of ultra-fast methods for virus identification based on sensor principles. We created a structure modeling surface and size of the SARS-CoV-2 virus and used it in comparison with the standard antigen SARS-CoV-2-the receptor-binding domain (RBD) of the S-protein of the envelope of the SARS-CoV-2 virus from the Wuhan strain-for the development of detection of coronaviruses using a DNA-modified, surface-enhanced Raman scattering (SERS)-based aptasensor in sandwich mode: a primary aptamer attached to the plasmonic surface-RBD-covered Ag nanoparticle-the Cy3-labeled secondary aptamer. Fabricated novel hybrid plasmonic structures based on "Ag mirror-SiO2-nanostructured Ag" demonstrate sensitivity for the detection of investigated analytes due to the combination of localized surface plasmons in nanostructured silver surface and the gap surface plasmons in a thin dielectric layer of SiO2 between silver layers. A specific SERS signal has been obtained from SERS-active compounds with RBD-specific DNA aptamers that selectively bind to the S protein of synthetic virion (dissociation constants of DNA-aptamer complexes with protein in the range of 10 nM). The purpose of the study is to systematically analyze the combination of components in an aptamer-based sandwich system. A developed virus size simulating silver particles adsorbed on an aptamer-coated sensor provided a signal different from free RBD. The data obtained are consistent with the theory of signal amplification depending on the distance of the active compound from the amplifying surface and the nature of such a compound. The ability to detect the target virus due to specific interaction with such DNA is quantitatively controlled by the degree of the quenching SERS signal from the labeled compound. Developed indicator sandwich-type systems demonstrate high stability. Such a platform does not require special permissions to work with viruses. Therefore, our approach creates the promising basis for fostering the practical application of ultra-fast, amplification-free methods for detecting coronaviruses based on SARS-CoV-2.

The monomeric C-reactive protein level is associated with the increase in carotid plaque number in patients with subclinical carotid atherosclerosis.

The high-sensitivity C-reactive protein (hsCRP) assay measures the level of the pentameric form of CRP in blood. Currently, there are no available assays measuring the level of the monomeric form of CRP (mCRP), produced at sites of local inflammation. We developed an assay measuring the mCRP level in blood plasma with functional beads for flow cytometry. The assay was used to measure the mCRP level in 80 middle-aged individuals with initially moderate cardiovascular SCORE risk. By the time of the mCRP measurement, the patients have been followed up for subclinical carotid atherosclerosis progression for 7 years. Ultrasound markers of subclinical atherosclerosis, which included plaque number (PN) and total plaque height (PH), were measured at baseline and at the 7th-year follow-up survey. Inflammatory biomarkers, including mCRP, hsCRP, inteleukin-6 (IL-6) and von Willebrand factor (VWF) level, were measured at the 7th-year follow-up survey. The median level of mCRP was 5.2 (3.3; 7.1) µg/L, hsCRP 1.05 (0.7; 2.1) mg/L, IL-6 0.0 (0.0; 2.8) pg/mL, VWF 106 (77; 151) IU/dL. In the patients with the mCRP level below median vs. the patients with the median mCRP level or higher, change from baseline in PN was 0.0 (0.0; 1.0) vs. 1.0 (1.0; 2.0) and PH 0.22 (-0.24; 1.91) mm vs. 1.97 (1.14; 3.14) mm, respectively (p < 0.05). The adjusted odds ratio for the formation of new carotid atherosclerotic plaques was 4.7 (95% CI 1.7; 13.2) for the patients with the median mCRP level or higher. The higher mCRP level is associated with the more pronounced increase in PN and PH in patients with normal level of traditional inflammatory biomarkers and initially moderate cardiovascular SCORE risk.

G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions.

G-quadruplexes (G4s) are a unique class of noncanonical DNAs that play a key role in cellular processes and neoplastic transformation. Herein, we focused on the promoter region of human TERT oncogene, whose product is responsible for the immortality of cancer cells. It has been shown by chemical probing and spectroscopic methods that synthetic 96-nt DNAs modeling the wild-type G-rich strand of the hTERT promoter and its variants with G>A point substitutions corresponding to somatic driver mutations fold into three stacked parallel G4s with sites of local G4 destabilization caused by G>A substitutions in the G4 motif. These models were used to elucidate how the hTERT multiG4 affects the binding affinity and functional responses of two key proteins, MutS and MutL, involved in the initial stage of DNA mismatch repair (MMR) in Escherichiacoli and Neisseriagonorrhoeae with different MMR mechanisms. We have shown for the first time that (i) point substitutions do not affect the effective binding of these proteins to the hTERT G4 structure, and (ii) the endonuclease activity of MutL from N. gonorrhoeae is significantly suppressed by the stable G4 scaffold. It is likely that some of the genomic instability associated with G4 may be related to the blockage of human intrinsic methyl-independent MMR attempting to operate near G4 structures.

Nanopore Sequencing for De Novo Bacterial Genome Assembly and Search for Single-Nucleotide Polymorphism.

Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an E. coli genome characterized by a deletion of the tolC gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Detection of TERT Promoter Mutations as a Prognostic Biomarker in Gliomas: Methodology, Prospects, and Advances.

This article reviews the existing approaches to determining the TERT promoter mutational status in patients with various tumoral diseases of the central nervous system. The operational characteristics of the most common methods and their transferability in medical practice for the selection or monitoring of personalized treatments based on the TERT status and other related molecular biomarkers in patients with the most common tumors, such as glioblastoma, oligodendroglioma, and astrocytoma, are compared. The inclusion of new molecular markers in the course of CNS clinical management requires their rapid and reliable assessment. Availability of molecular evaluation of gliomas facilitates timely decisions regarding patient follow-up with the selection of the most appropriate treatment protocols. Significant progress in the inclusion of molecular biomarkers for their subsequent clinical application has been made since 2016 when the WHO CNS classification first used molecular markers to classify gliomas. In this review, we consider the methodological approaches used to determine mutations in the promoter region of the TERT gene in tumors of the central nervous system. In addition to classical molecular genetical methods, other methods for determining TERT mutations based on mass spectrometry, magnetic resonance imaging, next-generation sequencing, and nanopore sequencing are reviewed with an assessment of advantages and disadvantages. Beyond that, noninvasive diagnostic methods based on the determination of the mutational status of the TERT promoter are discussed.

Telomere length regulation by Rif1 protein from Hansenula polymorpha.

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism - from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension - a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

Glycosylation of Receptor Binding Domain of SARS-CoV-2 S-Protein Influences on Binding to Immobilized DNA Aptamers.

Nucleic acid aptamers specific to S-protein and its receptor binding domain (RBD) of SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virions are of high interest as potential inhibitors of viral infection and recognizing elements in biosensors. Development of specific therapy and biosensors is complicated by an emergence of new viral strains bearing amino acid substitutions and probable differences in glycosylation sites. Here, we studied affinity of a set of aptamers to two Wuhan-type RBD of S-protein expressed in Chinese hamster ovary cell line and Pichia pastoris that differ in glycosylation patterns. The expression system for the RBD protein has significant effects, both on values of dissociation constants and relative efficacy of the aptamer binding. We propose glycosylation of the RBD as the main force for observed differences. Moreover, affinity of a several aptamers was affected by a site of biotinylation. Thus, the robustness of modified aptamers toward new virus variants should be carefully tested.

Urine TERT promoter mutations-based tumor DNA detection in patients with bladder cancer: A pilot study.

Telomerase reverse transcriptase (TERT) promoter mutations are the most frequent genetic events in bladder cancer (BC). The aim of the present pilot study was to evaluate the diagnostic potential of urine TERT promoter mutations-based liquid biopsy in patients with an ongoing oncological process, as well as in post-resection patients at risk of BC recurrence. A total of 60 patients were enrolled, of whom 27 patients had histologically proven BC; 23 had no signs of BC (control group); and 10 patients underwent transurethral malignancy resection 3-6 months prior to urine donation ('second look' group). Urine TERT promoter mutations were detected using Droplet Digital PCR. Receiver operating characteristic curve analysis revealed significant diagnostic power of the present approach (area under the curve: -0.768). At the cut-off value of tumor DNA fraction 0.34%, the sensitivity and specificity were 55.56 and 100%, respectively. In the positive samples, tumor DNA fraction varied significantly from 0.59 to 48.77%. In the 'second look' group, tumor DNA was detected in 4/10 patients, highlighting the possibility of BC recurrence with its fraction ranging only from 0.90 to 6.61%. Therefore, urine TERT promoter mutations-based liquid biopsy appears to be a promising tool for BC diagnosis and surveillance. The main study will include recruitment of additional patients, extension of the mutation panel, prolonged follow-up of the post-resection patients, as well as screening of industrial workers exposed to specific carcinogens.

Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair.

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.