Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action.

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses.

Emerging and re-emerging viruses periodically cause outbreaks and epidemics around the world, which ultimately lead to global events such as the COVID-19 pandemic. Thus, the urgent need for new antiviral drugs is obvious. Over more than a century of antiviral development, nucleoside analogs have proven to be promising agents against diversified DNA and RNA viruses. Here, we present the synthesis and evaluation of the antiviral activity of nucleoside analogs and their deglycosylated derivatives based on a hydroxybenzo[4,5]imidazo[1,2-c]pyrimidin-1(2H)-one scaffold. The antiviral activity was evaluated against a panel of structurally and phylogenetically diverse RNA and DNA viruses. The leader compound showed micromolar activity against representatives of the family Coronaviridae, including SARS-CoV-2, as well as against respiratory syncytial virus in a submicromolar range without noticeable toxicity for the host cells. Surprisingly, methylation of the aromatic hydroxyl group of the leader compound resulted in micromolar activity against the varicella-zoster virus without any significant impact on cell viability. The leader compound was shown to be a weak inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase. It also inhibited biocondensate formation important for SARS-CoV-2 replication. The active compounds may be considered as a good starting point for further structure optimization and mechanistic and preclinical studies.

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals.

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

Microarray Profiling of Vaccination-Induced Antibody Responses to SARS-CoV-2 Variants of Interest and Concern.

Mutations in surface proteins enable emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to escape a substantial fraction of neutralizing antibodies and may thus weaken vaccine-driven immunity. To compare available vaccines and justify revaccination, rapid evaluation of antibody (Ab) responses to currently circulating SARS-CoV-2 variants of interest (VOI) and concern (VOC) is needed. Here, we developed a multiplex protein microarray-based system for rapid profiling of anti-SARS-CoV-2 Ab levels in human sera. The microarray system was validated using sera samples from SARS-CoV-2-free donors and those diagnosed with COVID-19 based on PCR and enzyme immunoassays. Microarray-based profiling of vaccinated donors revealed a substantial difference in anti-VOC Ab levels elicited by the replication-deficient adenovirus vector-base (Sputnik V) and whole-virion (CoviVac Russia COVID-19) vaccines. Whole-virion vaccine-induced Abs showed minor but statistically significant cross-reactivity with the human blood coagulation factor 1 (fibrinogen) and thrombin. However, their effects on blood clotting were negligible, according to thrombin time tests, providing evidence against the concept of pronounced cross-reactivity-related side effects of the vaccine. Importantly, all samples were collected in the pre-Omicron period but showed noticeable responses to the receptor-binding domain (RBD) of the Omicron spike protein. Thus, using the new express Ab-profiling system, we confirmed the inter-variant cross-reactivity of the anti-SARS-CoV-2 Abs and demonstrated the relative potency of the vaccines against new VOCs.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring.

The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM-BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands.

DNA G-Quadruplexes (G4s) Modulate Epigenetic (Re)Programming and Chromatin Remodeling: Transient Genomic G4s Assist in the Establishment and Maintenance of Epigenetic Marks, While Persistent G4s May Erase Epigenetic Marks.

Here, the emerging data on DNA G-quadruplexes (G4s) as epigenetic modulators are reviewed and integrated. This concept has appeared and evolved substantially in recent years. First, persistent G4s (e.g., those stabilized by exogenous ligands) were linked to the loss of the histone code. More recently, transient G4s (i.e., those formed upon replication or transcription and unfolded rapidly by helicases) were implicated in CpG island methylation maintenance and de novo CpG methylation control. The most recent data indicate that there are direct interactions between G4s and chromatin remodeling factors. Finally, multiple findings support the indirect participation of G4s in chromatin reshaping via interactions with remodeling-related transcription factors (TFs) or damage responders. Here, the links between the above processes are analyzed; also, how further elucidation of these processes may stimulate the progress of epigenetic therapy is discussed, and the remaining open questions are highlighted.

DNA G-Quadruplexes Contribute to CTCF Recruitment.

G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.

Phenoxazine-based scaffold for designing G4-interacting agents.

G-quadruplexes (G4) represent one class of non-canonical secondary nucleic acid structures that are currently regarded as promising and attractive targets for anti-cancer, anti-viral and antibacterial therapy. Herein, we probe a new i-clamp-inspired phenoxazine scaffold for designing G4-stabilizing ligands. The length of the protonated aminoalkyl tethers ('arms') of the phenoxazine-based ligand was optimized in silico. Two double-armed ligands differing in the relative orientation of their arms and one single-armed ligand were synthesized. The two-armed ligands significantly enhanced the thermal stability of the G-quadruplex structures (increasing the melting temperature by up to 20 °C) and displayed G4 selectivity over duplex DNA. The ligands look promising for biological studies and the phenoxazine scaffold could be a starting point for designing new G4-interacting compounds.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability.

Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2'-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10-11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.

Polymorphism of G4 associates: from stacks to wires via interlocks.

We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues.

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA.

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.

Thiadiazole-, selenadiazole- and triazole-fused anthraquinones as G-quadruplex targeting anticancer compounds.

G-quadruplex (G4) ligands attract considerable attention as potential anticancer therapeutics. In this study we proposed an original scheme for synthesis of azole-fused anthraquinones and prepared a series of G4 ligands carrying amino- or guanidinoalkylamino side chains. The heterocyclic core and structure of the terminal groups strongly affect on binding to G4-forming oligonucleotides, cellular accumulation and antitumor potency of compounds. In particular, thiadiazole- and selenadiazole- but not triazole-based ligands inhibit the proliferation of tumor cells (e.g. K562 leukemia) and stabilize primarily telomeric and c-MYC G4s. Anthraselenadiazole derivative 11a showed a good affinity to c-MYC G4 in vitro and down-regulated expression of c-MYC oncogene in cellular conditions. Further studies revealed that anthraselenadiazole 11a provoked cell cycle arrest and apoptosis in a dose- and time-dependent manner inhibiting K562 cells growth. Taken together, this work gives a valuable example that the closely related heterocycles may cause a significant difference in biological properties of G4 ligands.

Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems.

New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.