3'-modified oligo (2'-O-methylribonucleotides) as improved probes for hybridization with RNA

A series of octa (2-O-methylribonucleotides) with an additional 3'-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3'-3' (inverted) bond was synthesized. The exceptional stability of these oligomers to a 3'-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3'-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2'-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal AmU(T) pair and enhances the mismatch sensitivity.     

RNase P-Guiding Peptide Conjugates of Oligo(2'-O-methylribonucleotides) as Prospective Antibacterial Agents

Russian Journal of Bioorganic Chemistry - A novel variant of the synthesis of 3'- and 5'-peptide conjugates of oligo(2'-O-methylribonucleotides) has been developed using thiol-maleimide...     

Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.     

Berberine derivatives as cationic fluorescent probes for the investigation of the energized state of mitochondria

The interaction of rat liver and bovine heart mitochondria with a series of fluorescent, cationic berberine derivatives varying in the length of alkyl chain has been investigated. An increase in the hydrophobicity of the derivative was accompanied by a larger value of the partition coefficient and by binding to a more hydrophobic region of the inner mitochondrial membrane. It was found that berberines could be used as sensitive indicators of processes which take place on the outer surface of the mitochondrial membrane; the greatest (15-fold) increase in fluorescence was obtained with 13-methylberberine in the energized state of mitochondria. The fluorescence increase was due to the increase in fluorescence quantum yield although a small increase in the amount of bound derivative could also be detected upon energization. The fluorescence was linearly dependent on the magnitude of the membrane potential. In parallel with an observed fluorescence enhancement a considerable decrease in rotational mobility was found. We suggest that berberines move in the inner membrane according to the polarity of the membrane potential; consequently, deeper immersion in the less polar region in the energized state brings about a larger fluorescence increase. More hydrophobic derivatives inhibited NAD-linked respiration in rat liver mitochondria but exerted no effect on succinate oxidation up to 10 μM concentration.     

7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Synthesis of fluorescent derivatives of wortmannin and demethoxyviridin as probes for phosphatidylinositol 3-kinase

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.     

Ligand reaction-based fluorescent peptide probes for the detection of Cu2+ and glutathione

Copper is a critical element in both human and animal metabolic processes. Its role includes supporting connective tissue cross-linking, as well as iron and lipid metabolism; at the same time, copper is also a toxic heavy metal that can cause harm to both the environment and human health. Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine combined with sulfhydryl groups. Its properties include acting as an antioxidant and facilitating integrative detoxification. GSH is present in both plant and animal cells and has a fundamental role in maintaining living organisms. GSH is the most abundant thiol antioxidant in the human body. It exists in reduced and oxidized forms within cells and provides significant biochemical functions, such as regulating vitamins such as vitamins D, E, and C, and facilitating detoxification. A fluorescent probe has been developed to detect copper ions selectively, sensitively, and rapidly. This report outlines the successful work on creating a peptide probe, TGN (TPE-Trp-Pro-Gly-Cln-His-NH₂), with specific Cu²⁺ detection capabilities, and a significant fluorescence recovery occurred with the addition of GSH. This indicates that the probe can detect Cu²⁺ and GSH concurrently. The detection limit for Cu²⁺ in the buffer solution was 264 nM (R² = 0.9992), and the detection limit for GSH using the TGN-Cu²⁺ complex was 919 nM (R² = 0.9917). The probe exhibits high cell permeability and low biotoxicity that make it ideal for live cell imaging in biological conditions. This peptide probe has the capability to detect Cu²⁺ and GSH in biological cells.     

A method for the detection and characterization of GABA(A) receptors by calcium-sensitive fluorescent probes

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.     

一种基于荧光qPCR检测新型冠状病毒核酸的优化反应体系的建立及相关测试

为实时并快速地检出SARS-CoV-2冠状病毒RNA,对基于荧光定量聚合酶链式反应(Polymerasechain reaction,PCR)的反应体系进行了优化。结果表明,按照本实验提供的方法操作后,检测SARS-CoV-2冠状病毒所用的RNA样本的最小浓度稀释度可调至1/10000(初始值设为10ng/μL)。且用于检测COVID-19临床阳性样本所测得循环值(Cycle threshold,Ct)均低于35或40。其灵敏性测试结果也表明该方法的敏感性较好。同时在同等条件下,与目前市场上的COVID-19试剂盒的检测结果基本一致,并且检测循环数缩短2个单位。因此,本实验所建立的体系适用于前期临床诊断的筛查工作,为在医学上实现快速诊断提供了工具。     

Synthesis of conjugated spermine derivatives with 7-nitrobenzoxadiazole (NBD), rhodamine and bodipy as new fluorescent probes for the polyamine transport system

The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N₁-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.     

Hybridization of Oligo(dT) to RNA on nitrocellulose

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.     

Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC₅₀ values of 18–100 nM in a standard TRAP assay.     

Characterization of a series of far-red-absorbing thiobarbituric acid oxonol derivatives as fluorescent probes for biological applications

Chromophores that absorb in the far-red region of the spectrum are increasingly being utilized for applications in the biosciences. We have synthesized and evaluated a novel series of fluorescent oxonols based on thiobarbituric acids containing aryl and heteroaryl substituents. The novel chromophores possess narrow absorption spectra ( approximately 40-nm bandwidths), reasonable Stokes shifts ( approximately 25 nm), and quantum yields of up to 0.67 in organic solvents and 0.3 in aqueous solvents, with absorption wavelength maxima at 620-640 nm. The spectral properties of the compounds are sensitive to base and exhibit a loss of far-red absorbance that is concentration and time dependent. Derivatives have been synthesized that can be used for the labeling of macromolecules such as proteins and DNA. The probes show environment sensitivity and the oligonucleotide conjugates sense the formation of duplex DNA. These novel far-red fluorophores have potential applications in diagnostic and research applications.     

A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca²⁺ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC₅₀ for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.     

Synthesis and evaluation of fluorescent probes for the detection of calpain activity

Two new probes for the detection of calpain I activity based on fluorescence resonance energy transfer technology have been synthesized and evaluated. The probes incorporated the cleavage site present in alpha-spectrin, a naturally occurring substrate of calpain I. The design of the internally quenched substrates is such that the calpain-sensitive bond of the peptides (between the Tyr-Gly residues) is located centrally between the donor and the quencher chromophores. The calpain assay protocol is capable of detecting enzymatic activity in the nanomolar region.     

Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells

An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.     

Pyrene derivatives as fluorescent probes of conformation near the 3′ termini of polyribonucleotides

When pyrene butyric acid hydrazide or pyrene acetic acid hydrazide is attached to single-strand RNA 3′ termini a red shift in absorbance and substantial hypochromicity are observed. A strong induced CD is seen and the fluorescence intensity is quenched by an order of magnitude. In double-stranded samples, a further 10-fold quenching of fluorescence is seen. Several lines of evidence suggest that the residual fluorescence of pyrene butyric acid hydrazide-duplex conjugates arises from a minor species. The most likely possibility is dye reacted at a site other than the 3′ end. Some indication exists that 3′-attached pyrene may perturb the relative stability of nearby duplex. Within the limits of this reservation, it appears that 3′-pyrene conjugates may be rather useful for detecting the existence of duplex regions accessible to a dye at the 3′ end of complex RNAs.