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Simple Summary: Apoptosis is one of the best-known types of programmed cell death. This process is regulated by a number of genes and proteins, among which the Bcl-2 protein family plays a key role. This family includes anti- and proapoptotic proteins. Cancer cell resistance to apoptosis is commonly associated with overexpression of the antiapoptotic members of Bcl-2 family proteins, in particular, Bcl-2, Bcl-xL, and Mcl-1. Subsequently, these proteins represent perspective targets for anticancer therapy. Here, using an inhibitory approach, we found that Bak and Bcl-xL regulate sensitivity of cancer cells to Mcl-1 inhibition. BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition. [ABSTRACT FROM AUTHOR]

We developed a novel robust technique to produce high-quality dispersions of debundled SWCNTs in aqueous solutions. Direct injection of SWCNTs treated with supercritical nitrogen into the aqueous surfactant solution facilitates the dispersion process without the need of extensive ultrasonication. According to photoluminescence and absorbance measurements, the mild ultrasonic treatment of such dispersions resulted in a higher yield of individual SWCNTs, compared to pristine tubes and tubes collected after the supercritical treatment in the form of powder. [ABSTRACT FROM AUTHOR]

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO 3 ) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO 3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.

Accurate monitoring of low levels of viral load (the number of viral particles per milliliter of plasma) in HIV-infected patients is important in terms of evaluation of the progress of antiretroviral therapy. The general approach for detection of low copy HIV RNA is reverse transcription combined with quantitative real-time PCR based on fluorescence detection. The selection of primers and the structure of fluorogenic oligonucleotide probes are crucial for sensitivity and accuracy of the assay. In this chapter, we report the RT-qPCR protocol for detection of low copy HIV RNA using double stranded Yin-Yang DNA probes containing identical fluorescent dyes on each strand of the probe. Dye residues attached to the 3'-end of an oligonucleotide and 5'-end of the complementary oligonucleotide form a self-quenched aggregate in a Yin-Yang duplex probe, and display fluorescence light up upon probe strand displacement with the target sequence amplified in the course of PCR. Among several fluorescent dyes tested (R6G, ROX, Cy5) the ROX labeled Yin-Yang probes showed better fluorescence increase and lower C t values. All the homo Yin-Yang probes were superior to corresponding dye-quencher probes and allowed reliable detection of 10-10,000 copies of HIV RNA per mL.

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G 8AE -clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.

We developed a metal ion chelate-assisted ligation for SNP detection by microarray. An oligonucleotide probe was separated into two 9-10-mers bearing iminodiacetic residues at the gap point. Duplex formation with the DNA target was possible only if nickel ions were added, but a nucleotide substitution opposite the gap point prevented duplex formation. Here we demonstrate the application of this approach for SNP detection (A1298C) within the 5,10-methylenetetrahydrofolate reductase gene on a microarray.

Excimer formation is a unique feature of some fluorescent dyes (e.g., pyrene) which can be used for probing the proximity of biomolecules. Pyrene excimer fluorescence has previously been used for homogeneous detection of single nucleotide polymorphism (SNP) on DNA. 1-Phenylethynylpyrene (1-1-PEPy), a photostable pyrene derivative with redshifted fluorescence, is able to form excimers (emission maximum about 500-510 nm) and is well suitable for nucleic acid labeling. We have shown the utility of 1-1-PEPy in the excimer-forming DNA probes for detection of 2144A/G and 2143A/G transitions, and 2143A/C substitution in the 23S ribosomal RNA gene of Helicobacter pylori strains resistant to clarithromycin. The phenylethynylpyrene pair can be generated either from 1-1-PEPy pseudonucleoside 4-[4-(pyren-1-ylethynyl)phenyl]-1,3-butanediol or from 2'-O-(1-PEPy) modified nucleosides--2'-O-[3-(pyren-1-ylethynyl)benzyl]uridine and 2'-O-[4-(pyren-1-ylethynyl)benzyl]uridine.

This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group addition-elimination reactions in nucleic acids chemistry are discussed.

2'-Deoxyoligonucleotides and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy, or hydrazide group to give peptide-oligonucleotide conjugates incorporating single or multiple peptides in good yield. The facile conjugation method allows specific coupling in aqueous solution of unprotected oligonucleotides containing aldehyde groups to unprotected N-terminally modified peptides and other small molecules. A 12-mer 2'-O-methyloligoribonucleotide complementary to the HIV-1 TAR RNA stem-loop and containing two conjugated copies of an 8-mer model laminin peptide was hardly affected in TAR RNA binding and showed a similar level of inhibition of HIV-1 Tat-dependent in vitro transcription compared to the unconjugated 2'-O-methyloligoribonucleotide. Advantages of this conjugation method include (1) the ability to attach more than one peptide or other small molecule to oligonucleotide at defined nucleoside residue locations; (2) a conjugation route that does not affect significantly oligonucleotide binding to RNA structures; and (3) three alternative, facile, and mild conjugation reaction types that do not require use of a large excess of peptide reagent.