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Bruce A Armitage - One of the best experts on this subject based on the ideXlab platform.
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strand invasion of mixed sequence b dna by acridine linked γ peptide nucleic acid γ pna
Journal of the American Chemical Society, 2007Co-Authors: Srinivas Rapireddy, Subhadeep Roy, Bruce A ArmitageAbstract:Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson−Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized γ-peptide nucleic acid (γ-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson−Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.
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strand invasion of mixed sequence b dna by acridine linked gamma peptide nucleic acid gamma pna
Journal of the American Chemical Society, 2007Co-Authors: Srinivas Rapireddy, Subhadeep Roy, Bruce A ArmitageAbstract:Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson−Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized γ-peptide nucleic acid (γ-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson−Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.
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recent advances in the development of peptide nucleic acid as a gene targeted drug
Expert Opinion on Biological Therapy, 2004Co-Authors: Violeta L Marin, Subhadeep Roy, Bruce A ArmitageAbstract:Peptide nucleic acid (PNA) is a non-ionic mimic of DNA that binds to complementary DNA and RNA sequences with high affinity and selectivity. Targeting of single-stranded RNA leads to antisense effects, whereas PNAs directed toward double-stranded DNA exhibit antigene properties. Recent advances in cell uptake and in antisense and antigene effects in biological systems are summarised in this review. In addition to traditional targets, namely genomic DNA and messenger RNA, applications for PNA as a bacteriocidal antibiotic, for regulating splice site selection and as a telomerase inhibitor are described.
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the impact of nucleic acid secondary structure on pna hybridization
Drug Discovery Today, 2003Co-Authors: Bruce A ArmitageAbstract:Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.
Masad J Damha - One of the best experts on this subject based on the ideXlab platform.
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studies on the hydrolytic stability of 2 fluoroarabinonucleic acid 2 f ana
Organic and Biomolecular Chemistry, 2009Co-Authors: Jonathan K Watts, Adam Katolik, Julia Viladoms, Masad J DamhaAbstract:The stability of 2′-deoxy-2′-fluoroarabinonucleic acid (2′F-ANA) to hydrolysis under acidic and basic conditions was compared to that of DNA, RNA and 2′F-RNA. In enzyme-free simulated gastric fluid (pH ∼1.2), 2′F-ANA was found to have dramatically increased stability (virtually no cleavage observed after 2 days) with respect to both DNA (t1/2∼ 2 min) and RNA (t1/2∼ 3 h (PO) or 3 days (PS)). These results were observed for both phosphodiester and phosphorothioate backbones and with multiple mixed-base sequences. Under basic conditions, 2′F-ANA also showed good stability. In 1 M NaOH at 65 °C, 2′F-ANA had a t1/2 of ∼20 h, while RNA was entirely degraded in a few minutes. Furthermore, the nuclease cleavage of phosphorothioate 2′F-ANA and DNA by snake venom phosphodiesterase was studied in detail. One diastereomer of the PS-2′F-ANA linkage was found to be much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. Additional studies of 2′F-ANA-containing oligonucleotides are warranted based on the excellent stability properties described here.
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duplex recognition by oligonucleotides containing 2 deoxy 2 fluoro d arabinose and 2 deoxy 2 fluoro d ribose intermolecular 2 oh phosphate contacts versus sugar puckering in the stabilization of triple helical complexes
Bioconjugate Chemistry, 1999Co-Authors: Christopher J Wilds, Masad J DamhaAbstract:To gain insight into the origins of the large binding affinity of RNA toward target duplexes, 2‘-deoxy-2‘-fluororibonucleic acid (2‘F-RNA) and 2‘-deoxy-2‘-fluoroarabinonucleic acid (2‘F-ANA) were tested for their ability to recognize duplex DNA, duplex RNA, and RNA−DNA hybrids. 2‘F-RNA, 2‘F-ANA, and the corresponding control single-stranded (ss) DNA strands were shown to form triple-helical complexes only with duplex DNA and hybrid DNA (Pu)−RNA (Py), but not with duplex RNA and hybrid RNA (Pu)−DNA (Py). In contrast, an RNA third strand recognized all four possible duplexes (DD, DR, RD, and RR) as previously demonstrated by Roberts and Crothers [(1992) Science 258, 1463−1466]. The 2‘F-RNA (C3‘-endo) strand exhibited significantly reduced affinity for duplexes compared to an unmodified RNA (C3‘-endo) strand. These findings are consistent with the intermolecular 2‘-OH−phosphate contact mechanism proposed by Escude et al. [(1993) Nucleic Acids Res. 24, 5547−5553], as a ribo 2‘-F atom should not interact with ...
Cordula Haas - One of the best experts on this subject based on the ideXlab platform.
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RNA dna co analysis from human menstrual blood and vaginal secretion stains results of a fourth and fifth collaborative ednap exercise
Forensic Science International-genetics, 2014Co-Authors: Cordula Haas, Erin K. Hanson, M.j. Anjos, Kaye N. Ballantyne, R. Banemann, Bryan Bhoelai, Eduardo Euclydes De Lima E Borges, Mónica Carvalho, Cornelius Courts, G. De CockAbstract:Abstract The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4–1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.
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RNA dna co analysis from human saliva and semen stains results of a third collaborative ednap exercise
Forensic Science International-genetics, 2013Co-Authors: Cordula Haas, Erin K. Hanson, M.j. Anjos, R. Banemann, Eduardo Euclydes De Lima E Borges, Mónica Carvalho, Cornelius Courts, Andrea Berti, Angel Carracedo, G. De CockAbstract:A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 μl saliva, 5-0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.
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capillary electrophoresis of a multiplex reverse transcription polymerase chain reaction to target messenger RNA markers for body fluid identification
Methods of Molecular Biology, 2012Co-Authors: Cordula Haas, Erin K. Hanson, Jack BallantyneAbstract:Abstract The analysis of cell-specific mRNA expression is a promising new method for the identification of body fluids. A number of mRNA markers have been identified for the forensically most relevant body fluids: blood, saliva, semen, vaginal secretions, and menstrual blood. Apart from a significant improvement in specificity compared to conventional protein-based methods, other important advantages of body fluid identification by mRNA profiling include the possibility of simultaneously isolating RNA and DNA from the same piece of stain and the ability to multiplex numerous RNA markers for the identification of one or several body fluids. RNA profiling can be incorporated into current DNA analysis pipelines.
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collaborative ednap exercises on messenger RNA dna co analysis for body fluid identification blood saliva semen and str profiling
Forensic Science International: Genetics Supplement Series, 2011Co-Authors: Cordula Haas, Erin K. Hanson, Niels Morling, Jack BallantyneAbstract:RNA profiling is a new method for the identification of forensically relevant biological stains, such as blood, saliva, semen, vaginal secretions, menstrual blood, sweat and skin. To demonstrate the suitability of mRNA profiling for use in forensic casework, three collaborative exercises on RNA analysis or RNA/DNA co-analysis for body fluid identification (blood, saliva, semen) and STR profiling were organized within the European DNA Profiling Group (EDNAP). The results of these collaborative exercises support the potential use of an mRNA-based system for the identification of body fluids along with conventional DNA profiling.
Satoshi Obika - One of the best experts on this subject based on the ideXlab platform.
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dna polymerase variants with high processivity and accuracy for encoding and decoding locked nucleic acid sequences
Journal of the American Chemical Society, 2020Co-Authors: Hidekazu Hoshino, Yuuya Kasahara, Masayasu Kuwahara, Satoshi ObikaAbstract:Xenobiotic nucleic acids (XNAs) are chemically modified nucleic acid analogues with potential applications in nucleic acid-based therapeutics including nucleic acid aptamers, ribozymes, small interfering RNAs, and antisense oligonucleotides. We have developed a promising XNA for therapeutic uses, 2',4'-bridged nucleic acid (2',4'-BNA), also known as locked nucleic acid (LNA). Unlike the rational design of small interfering and antisense oligonucleotides, the development of LNA aptamers and catalysts requires genetically engineered polymerases that enable the synthesis of LNA from DNA and the converse reverse transcription. However, no LNA decoders or encoders with sufficient performance have been developed. In this study, we developed variants of KOD DNA polymerase, a family B DNA polymerase derived from Thermococcus kodakarensis KOD1, which are effective LNA decoders and encoders, via structural analyses. KOD DGLNK (KOD: N210D/Y409G/A485L/D614N/E664K) enabled LNA synthesis from DNA (DNA → LNA), and KOD DLK (KOD: N210D/A485L/E664K) enabled LNA reverse transcription to DNA (LNA → DNA). Both variants exhibited greatly improved efficiency and accuracy. Notably, we synthesized LNAs longer than one kilobase using KOD DGLNK. We also showed that these variants can accept 2'-O-methyl (2'-OMe), a common modification for therapeutic uses. Here, we also show that LNA and 2'-OMe mix aptamer can be practically obtained via SELEX. The variants can be used as powerful tools for creating XNA aptamers and catalysts to completely eliminate the natural species, DNA and RNA.
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synthesis and thermal stabilities of oligonucleotides containing 2 o 4 c methylene bridged nucleic acid with a phenoxazine base
Organic and Biomolecular Chemistry, 2017Co-Authors: Yuki Kishimoto, Yoshiyuki Hari, Akane Fujii, Osamu Nakagawa, Takanori Yokota, Tetsuya Nagata, Satoshi ObikaAbstract:We designed and synthesized a novel artificial 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2′,4′-BNA/LNA with 5-methylcytosine or 2′-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2′,4′-BNA/LNA moiety in DNA/RNA duplexes.
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Synthesis and Properties of 7‑Deazapurine- and 8‑Aza-7-deazapurine-Locked Nucleic Acid Analogues: Effect of the Glycosidic Torsion Angle
2016Co-Authors: Takashi Hara, Tetsuya Kodama, Yumi Takegaki, Kunihiko Morihiro, Kosuke Ramon Ito, Satoshi ObikaAbstract:Conformationally restricted nucleoside analogues 2′,4′-BNA/LNA-7-deazaguanine (LNA-7cG) and 2′,4′-BNA/LNA-8-aza-7-deazaguanine (LNA-8n7cG), which avoid extra hydrogen bond formation at the 7-position of the guanine nucleobase, were successfully synthesized and incorporated into oligonucleotides. While the LNA-7cG-containing oligonucleotides show high duplex-forming ability with complementary DNA and RNA similar to LNA-G, the LNA-8n7cG-containing oligonucleotide has lower binding affinity than that of natural 2′-deoxyguanosine. This disparity in thermostability is also observed in 7-deazaadenosine analogues (LNA-7cA, LNA-8n7cA). Thermodynamic parameters and computational chemistry revealed that an inappropriate glycosidic torsion angle χ of 2′,4′-BNA/LNA-8-aza-7-deazapurine analogues destabilizes duplex formation in contrast to 2′,4′-BNA/LNA-7-deazapurine analogues. This result indicates that the nucleobase rotation angle plays an important role in duplex binding affinity. In addition, LNA-7cG-modified oligonucleotide effectively suppresses aggregation even in a guanine-rich sequence
Stephan Vagner - One of the best experts on this subject based on the ideXlab platform.
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the g quadruplex specific RNA helicase dhx36 regulates p53 pre mRNA 3 end processing following uv induced dna damage
Journal of Molecular Biology, 2017Co-Authors: Michelle Newman, Rym Sfaxi, Abhijit Saha, David Monchaud, Mariepaule Teuladefichou, Stephan VagnerAbstract:Pre-mRNA 3 '-end processing, the process through which almost all eukaryotic mRNAs acquire a poly(A) tail is generally inhibited during the cellular DNA damage response leading to a profound impact on the level of protein expression since unprocessed transcripts at the 3 '-end will be degraded or unable to be transported to the cytoplasm. However, a compensatory mechanism involving the binding of the hnRNP H/F family of RNA binding proteins to an RNA G-quadruplex (G4) structure located in the vicinity of a polyadenylation site has previously been described to allow the transcript encoding the p53 tumour suppressor protein to be properly processed during DNA damage and to provide the cells with a way to react to DNA damage. Here we report that the DEAH (Asp-Glu-Ala-His) box RNA helicase DHX36/RHAU/G4R1, which specifically binds to and resolves parallel-stranded G4, is necessary to maintain p53 pre-mRNA 3 '-end processing following UV-induced DNA damage. DHX36 binds to the p53 RNA G4, while mutation of the G4 impairs the ability of DHX36 to maintain pre-mRNA 3 '-end processing. Stabilization of the p53 RNA G4 with two different G4 ligands ((PNA)DOTASQ and PhenDC3), which is expected from previous studies to prevent DHX36 from binding and unwinding G4s, also impairs p53 pre-mRNA 3 '-end processing following UV. Our work identifies DHX36 as a new actor in the compensatory mechanisms that are in place to ensure that the mRNAs encoding p53 are still processed following UV. (C) 2016 Elsevier Ltd. All rights reserved.
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dna damage RNA binding proteins protect from near and far
Trends in Biochemical Sciences, 2014Co-Authors: Stephan Vagner, Martin Dutertre, Sarah Lambert, Aura Carreira, Mounira AmorgueretAbstract:Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the prevention of genome instability. These studies show that RBPs prevent harmful RNA/DNA hybrids and are involved in the DNA damage response (DDR), from DNA repair to cell survival decisions. Indeed, specific RBPs allow the selective regulation of DDR genes at multiple post-transcriptional levels (from pre-mRNA splicing/polyadenylation to mRNA stability/translation) and are directly involved in DNA repair. These multiple activities are mediated by RBP binding to mRNAs, nascent transcripts, noncoding RNAs, and damaged DNA. Finally, because DNA damage modifies RBP localization and binding to different RNA/DNA molecules, we propose that upon DNA damage, RBPs coordinately regulate various aspects of both RNA and DNA metabolism.
