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Jesper Wengel - One of the best experts on this subject based on the ideXlab platform.
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Application of Locked Nucleic Acid-based probes in fluorescence in situ hybridization
Applied microbiology and biotechnology, 2016Co-Authors: Silvia Fontenete, Jesper Wengel, Daniel T. O. Carvalho, Nuno Guimarães, Pedro Madureira, Ceu Figueiredo, Nuno F. AzevedoAbstract:Fluorescence in situ hybridization (FISH) employing Nucleic Acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that Locked Nucleic Acid (LNA) and 2′-O-methyl (2′-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of Nucleic Acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2′-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.
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Designation and sequence of the oligonucleotide probes containing Locked Nucleic Acid (LNA; with L superscript) and 2’-O-methyl RNA (2’-OMe; in Boldface) nucleotide monomers.
2015Co-Authors: Silvia Fontenete, Jesper Wengel, Nuno Guimarães, Pedro Madureira, Ceu Figueiredo, Marina Leite, Rui Manuel Ferreira, Nuno Filipe AzevedoAbstract:HP_ LNA/2OMe _PS is a phosphorothioate oligomer (PS backbones) labeled with either FAM (Fluorescein) or Cy3 (Cyanine).Designation and sequence of the oligonucleotide probes containing Locked Nucleic Acid (LNA; with L superscript) and 2’-O-methyl RNA (2’-OMe; in Boldface) nucleotide monomers.
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a Locked Nucleic Acid based nanocrawler designed and reversible movement detected by multicolor fluorescence
Journal of the American Chemical Society, 2013Co-Authors: Kira I Astakhova, Karol Pasternak, Meghan A Campbell, Pankaj Gupta, Jesper WengelAbstract:Herein we introduce a novel fluorescent LNA/DNA machine, a nanocrawler, which reversibly moves along a directionally polar complementary road controlled by affinity-enhancing Locked Nucleic Acid (LNA) monomers and additional regulatory strands. Polyaromatic hydrocarbon (PAH) dyes attached to 2′-amino-LNA monomers are incorporated at four stations of the system, enabling simple detection of the position of the nanocrawler via a step-specific color signal. The sensing is provided by highly sensitive, chemically stable, and photostable PAH LNA interstrand communication systems, including pyrene excimer formation and pyrene–perylene interstrand Forster resonance energy transfer. We furthermore demonstrate that the nanocrawler selectively and reversibly moves along the road, followed by a bright and consistent fluorescence response for up to 10 cycles without any loss of signal.
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Cleavage and protection of Locked Nucleic Acid-modified DNA by restriction endonucleases.
Bioorganic & medicinal chemistry letters, 2012Co-Authors: Lucile Crouzier, Jesper Wengel, Camille Dubois, Rakesh N. VeeduAbstract:Locked Nucleic Acid (LNA) is one of the most prominent Nucleic Acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.
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efficient reverse transcription using Locked Nucleic Acid nucleotides towards the evolution of nuclease resistant rna aptamers
PLOS ONE, 2012Co-Authors: Lucile Crouzier, Jesper Wengel, Camille Dubois, Stacey L Edwards, Lasse Holm Lauridsen, Rakesh N. VeeduAbstract:Background Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked Nucleic Acid (LNA) is one of the most prominent and successful Nucleic Acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection. Methodology In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition. Conclusions We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers
Rakesh N. Veedu - One of the best experts on this subject based on the ideXlab platform.
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Rational Design of Short Locked Nucleic Acid-Modified 2′-O-Methyl Antisense Oligonucleotides for Efficient Exon-Skipping In Vitro
Elsevier, 2017Co-Authors: Abbie M. Adams, Susan Fletcher, Stephen D. Wilton, Rakesh N. VeeduAbstract:Locked Nucleic Acid is a prominent Nucleic Acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of Locked Nucleic Acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated Locked Nucleic Acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing Locked Nucleic Acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs
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Cleavage and protection of Locked Nucleic Acid-modified DNA by restriction endonucleases.
Bioorganic & medicinal chemistry letters, 2012Co-Authors: Lucile Crouzier, Jesper Wengel, Camille Dubois, Rakesh N. VeeduAbstract:Locked Nucleic Acid (LNA) is one of the most prominent Nucleic Acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.
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efficient reverse transcription using Locked Nucleic Acid nucleotides towards the evolution of nuclease resistant rna aptamers
PLOS ONE, 2012Co-Authors: Lucile Crouzier, Jesper Wengel, Camille Dubois, Stacey L Edwards, Lasse Holm Lauridsen, Rakesh N. VeeduAbstract:Background Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked Nucleic Acid (LNA) is one of the most prominent and successful Nucleic Acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection. Methodology In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition. Conclusions We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers
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Locked Nucleic Acid as a novel class of therapeutic agents
RNA biology, 2009Co-Authors: Rakesh N. Veedu, Jesper WengelAbstract:Locked Nucleic Acid (LNA) is a Nucleic Acid analogue with unprecedented binding affinity and excellent specificity toward complementary RNA and DNA oligonucleotides. The remarkable properties of LNA have led to applications within various gene silencing strategies both in vitro and in vivo. In the present review, we highlight the uses of LNA for regulation of gene expression with emphasis on RNA targeting.
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Locked Nucleic Acid nucleoside triphosphates and polymerases: on the way towards evolution of LNA aptamers.
Molecular bioSystems, 2009Co-Authors: Rakesh N. Veedu, Jesper WengelAbstract:Among numerous Nucleic Acid analogs reported in the past decades, Locked Nucleic Acid (LNA) has received substantial attention and has become a significant tool within chemical biology disciplines like molecular biology research, diagnostics and therapeutic development. However, despite their obvious structurally unique properties, LNA-based aptamers for diagnostic and therapeutic applications remain largely unexplored. Future evolution of LNAoligonucleotideaptamers will depend on scientific breakthroughs relating to enzymatic polymerization using LNAnucleoside triphosphates as substrates. Herein, we highlight recent developments in this direction using various polymerases.
David R Corey - One of the best experts on this subject based on the ideXlab platform.
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implications of high affinity hybridization by Locked Nucleic Acid oligomers for inhibition of human telomerase
Biochemistry, 2002Co-Authors: Anissa N Elayadi, Dwaine A Braasch, David R CoreyAbstract:Oligonucleotides that contain Locked Nucleic Acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of Nucleic Acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide Nucleic Acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.
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implications of high affinity hybridization by Locked Nucleic Acid oligomers for inhibition of human telomerase
Biochemistry, 2002Co-Authors: Anissa N Elayadi, Dwaine A Braasch, David R CoreyAbstract:Oligonucleotides that contain Locked Nucleic Acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of Nucleic Acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucl...
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Locked Nucleic Acid lna fine tuning the recognition of dna and rna
Chemistry & Biology, 2001Co-Authors: Dwaine A Braasch, David R CoreyAbstract:Abstract Locked Nucleic Acid is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2′-oxygen and the 4′-carbon. This conformation restriction increases binding affinity for complementarity sequences and provides an exciting new chemical approach for the control of gene expression and optimization of microarrays.
Weihong Tan - One of the best experts on this subject based on the ideXlab platform.
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Locked Nucleic Acid based beacons for surface interaction studies and biosensor development
Analytical chemistry, 2009Co-Authors: Karen Martinez, Colin D. Medley, M.-carmen Estevez, Joseph A. Phillips, Weihong TanAbstract:DNA sensors and microarrays permit fast, simple and real-time detection of Nucleic Acids through the design and use of increasingly sensitive, selective and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon’s poor stability due to the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds Locked Nucleic Acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the Locked Nucleic Acid bases for Nucleic Acid design for surface immobilization.
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synthesis and investigation of deoxyriboNucleic Acid Locked Nucleic Acid chimeric molecular beacons
Nucleic Acids Research, 2007Co-Authors: Chaoyong James Yang, Lin Wang, Colin D. Medley, Youngmi Kim, Hui Lin, Weihong TanAbstract:To take full advantage of Locked Nucleic Acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.
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Locked Nucleic Acid molecular beacons for intracellular mRNA monitoring
Nanobiophotonics and Biomedical Applications III, 2006Co-Authors: Lin Wang, Chaoyong James Yang, Colin D. Medley, Steven A. Benner, Weihong TanAbstract:We have designed, synthesized, and investigated a novel molecular beacon (MB) using Locked Nucleic Acid (LNA) bases for intracellular mRNA monitoring. This new LNA-MB has several useful properties including: very high melting temperature; excellent affinity for complementary sequences; superior single base mismatch discrimination capability; stablity against nuclease digestion; and not binding with single-stranded DNA binding proteins. All of these properties are highly advantageous for a molecular tool for various intracellular studies of biochemical, biological and medical significance.
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Locked Nucleic Acid molecular beacons.
Journal of the American Chemical Society, 2005Co-Authors: Lin Wang, Chaoyong James Yang, Colin D. Medley, Steven A. Benner, Weihong TanAbstract:A novel LNA-MB (molecular beacon based on Locked Nucleic Acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.
Ülo Langel - One of the best experts on this subject based on the ideXlab platform.
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Splice switching efficiency and specificity for oligonucleotides with Locked Nucleic Acid monomers
Biochemical Journal, 2008Co-Authors: Peter Guterstam, Jesper Wengel, Maria Lindgren, Henrik J Johansson, Ulf Tedebark, Samir El-andaloussi, Ülo LangelAbstract:The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced β-globin intron, HeLa pLuc705, were used to monitor splice switching activity of modified oligonucleotides by detection of expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing Locked Nucleic Acid monomers provide outstanding splice switching activity. However, similar oligonucleotides with several mismatches do not impede splice switching activity which indicates risk for off-target effects. The splice switching activity is abolished when mismatches are introduced at several positions with Locked Nucleic Acid monomers suggesting that it is the Locked Nucleic Acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice switching oligonucleotides and suggest that splice switching activity is tunable by utilizing Locked Nucleic Acid monomers.
