G-quadruplex induced stabilization by 2′-deoxy-2′-fluoro-d-arabinonucleic acids (2′F-ANA)

The impact of 2′-deoxy-2′-fluoroarabinonucleotide residues (2′F-araN) on different G-quadruplexes derived from a thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), an anti-HIV phosphorothioate aptamer PS-d(T2G4T2) and a DNA telomeric sequence d(G4T4G4) via UV thermal melting (Tm) and circular dichroism (CD) experiments has been investigated. Generally, replacement of deoxyguanosines that adopt the anti conformation (anti-guanines) with 2′F-araG can stabilize G-quartets and maintain the quadruplex conformation, while replacement of syn-guanines with 2′F-araG is not favored and results in a dramatic switch to an alternative quadruplex conformation. It was found that incorporation of 2′F-araG or T residues into a thrombin-binding DNA G-quadruplex stabilizes the complex (ΔTm up to ∼+3°C/2′F-araN modification); 2′F-araN units also increased the half-life in 10% fetal bovine serum (FBS) up to 48-fold. Two modified thrombin-binding aptamers (PG13 and PG14) show an approximately 4-fold increase in binding affinity to thrombin, as assessed via a nitrocellulose filter binding assay, both with increased thermal stability (∼1°C/2′F-ANA modification increase in Tm) and nuclease resistance (4–7-fold) as well. Therefore, the 2′-deoxy-2′-fluoro-d-arabinonucleic acid (2′F-ANA) modification is well suited to tune (and improve) the physicochemical and biological properties of naturally occurring DNA G-quartets

Synthesis and hybridization studies of oligonucleotides containing 1-(2-deoxy-2-α-C-hydroxymethyl-β-d-ribofuranosyl)thymine (2′-α-hm-dT)

We report the first investigation of oligoribonucleotides containing a few 1-(2-deoxy-2-α-C-hydroxymethyl-β-d-ribofuranosyl)thymine units (or 2′-hm-dT, abbreviated in this work as ‘H’). Both the 2′-CH(2)O-phosphoramidite and 3′-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2′,5′-RNA and RNA chains. The hybridization properties of the modified oligonucleotides have been studied via thermal denaturation and circular dichroism studies. While 3′,5′-linked H was shown previously to significantly destabilize DNA:RNA hybrids and DNA:DNA duplexes (modification in the DNA strand; ΔT(m) ∼ −3°C/insert), we find that 2′,5′-linked H have a smaller effect on 2′,5′-RNA:RNA and RNA:RNA duplexes (ΔT(m) = −0.3°C and −1.2°C, respectively). The incorporation of 3′,5′-linked H into 2′,5′-RNA:RNA and RNA:RNA duplexes was found to be more destabilizing (−0.7°C and −3.6°C, respectively). Significantly, however, the 2′,5′-linked H units confer marked stability to RNA hairpins when they are incorporated into a 2′,5′-linked tetraloop structure (ΔT(m) = +1.5°C/insert). These results are rationalized in terms of the compact and extended conformations of nucleotides

Structure–function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2′-5′ phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Δdbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure–activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching

Solid‐Phase Synthesis of Branched Oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143634/1/cpnc0414.pd

Stabilization of i-motif structures by 2'-β-fluorination of DNA

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology.Funding for open access charge: NSERC Discovery grant (to M.J.D., A.K.M.); CIHR DDTP Training Grant (to H.A., R.H.V.); MINECO [BFU2014-52864-R to C.G.]; CSIC-JAE contract (to N.M.P.).Peer Reviewe

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology

Effectiveness verification of an educational program about hearing protection for noise-exposed workers

OBJETIVO: Verificar a efetividade de uma ação educativa de treinamento, com ênfase na importância da proteção auditiva, para trabalhadores expostos a ruído ocupacional. MÉTODOS: Participaram 78 funcionários do gênero masculino. Todos os indivíduos passaram por avaliação audiológica completa e responderam a um questionário no momento do início do atendimento. Para a segunda aplicação do questionário, os participantes foram randomicamente divididos em dois grupos: Grupo Pesquisa, constituído por 44 funcionários, que responderam ao questionário após passarem por treinamento educativo, e Grupo Controle, constituído por 34 funcionários, que responderem ao questionário antes de passar por treinamento educativo. O treinamento foi feito com base em material gráfico com figuras e textos, sob a forma de conversa. Os temas abordados foram: importância da audição, efeitos do ruído sobre a saúde, importância da prevenção da perda auditiva e da utilização do protetor auditivo, conservação e higienização dos protetores, níveis de ruído no ambiente de trabalho e atenuação do ruído fornecida pelos protetores auditivos. O questionário continha 14 perguntas de múltipla escolha que abordavam os mesmos temas explorados no treinamento educativo. RESULTADOS: Houve aumento significativo do número de acertos durante a 2ª aplicação do questionário, somente para o Grupo Pesquisa, em todas as comparações realizadas. CONCLUSÃO: Ações educativas realizadas com trabalhadores expostos a ruído ocupacional são efetivas. Além disso, o questionário é uma ferramenta estável e viável para a verificação da efetividade de programas educativosPURPOSE: To verify the effectiveness of an educational action in the form of training, emphasizing the importance of hearing protection for workers exposed to occupational noise. METHODS: The study included 78 male individuals. All participants answered a questionnaire before they were submitted to audiological evaluation. For the second application of the questionnaire, participants were randomly divided into two groups: Research Group, constituted by 44 subjects that received educational training before the second questionnaire application, and Control Group, comprising 34 individuals that answered the questionnaire before the educational training. Training was based on material with graphic images and text, in the form of conversation. The topics covered included: the importance of hearing, noise effects on health, importance of preventing hearing loss and using hearing protection, conservation and cleaning of hearing protectors, levels of noise in the workplace and noise attenuation provided by hearing protectors. The questionnaire contained 14 multiple choice questions that addressed the same themes explored in the educational training. RESULTS: There was a significant increase of correct responses in the second application of the questionnaire, only in the Research Group, in all comparisons. CONCLUSION: Educational action performed with workers exposed to occupational noise are effective, and the questionnaire is a stable and viable tool to evaluate the effectiveness of educational program

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H

Stabilization of i-motif structures by 2'-β-fluorination of DNA

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology.This work is dedicated to the Memory of Alfredo Villasante, valuable collaborator and friend. FUNDING Funding for open access charge: NSERC Discovery grant (to M.J.D., A.K.M.); CIHR DDTP Training Grant (to H.A., R.H.V.); MINECO [BFU2014-52864-R to C.G.]; CSIC-JAE contract (to N.M.P.). Conflict of interest statement. None declaredS