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Indian journal of chemistry 01/2009; 48B:1709-1711.
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ABSTRACT: Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting.
The Journal of Organic Chemistry 01/2009; 74(3):1070-81. · 4.45 Impact Factor
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ABSTRACT: In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.
Chemistry 11/2008; 14(35):11010-26. · 5.93 Impact Factor
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ABSTRACT: This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.
FEBS Letters 10/2008; 582(20):3061-6. · 3.54 Impact Factor
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ABSTRACT: During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in vitro and in vivo. Herein, the synthesis of 2'- N-(perylen-3-yl)carbonyl-2'-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied 'zipper' motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields Phi F = 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to Phi F increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2'-position of 2'-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in vivo providing excitation wavelength, which completely eliminates cell autofluorescence.
Bioconjugate Chemistry 10/2008; 19(10):1995-2007. · 4.93 Impact Factor
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ABSTRACT: Polymerase chain reaction amplification of a locked nucleic acid (LNA)-modified DNA strand and transcription reactions using LNA-A nucleoside 5'-triphosphate were successfully accomplished with DNA and RNA polymerases.
Journal of the American Chemical Society 08/2008; 130(26):8124-5. · 9.91 Impact Factor
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Birte Vester,
Anne Marie Boel,
Sune Lobedanz,
B Ravindra Babu,
Michael Raunkjaer,
Dorthe Lindegaard,
Raunak,
Patrick J Hrdlicka,
Torben Højland,
Pawan K Sharma,
Surender Kumar,
Poul Nielsen, Jesper Wengel
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ABSTRACT: Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.
Bioorganic & medicinal chemistry letters 05/2008; 18(7):2296-300. · 2.65 Impact Factor
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ABSTRACT: Chemically modified nucleotide monomers were incorporated into adjacent terminal positions of two separate oligonucleotides complementary to an RNA target; all possible combinations of the catalytic units were tested, resulting in an artificial nuclease that showed high activity and catalytic turnover.
Chemical Communications 03/2008; · 6.17 Impact Factor
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ABSTRACT: Detection of nucleic acid hybridization via fluorescence resonance energy transfer (FRET) using pyren-1-ylmethyl and perylen-3-ylmethyl N2'-functionalized 2'-amino-LNA nucleosides incorporated into oligonucleotides exhibited a clear distance dependence of the FRET efficiency, ranging from below 10% when the fluorophores were approximately 40A apart to approximately 90% when the fluorophores were in close proximity.
Bioorganic & medicinal chemistry 02/2008; 16(1):94-9. · 2.82 Impact Factor
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ABSTRACT: A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue exhibiting enhanced hybridization efficiency toward complementary strand. The potential of LNA-based oligonucleotides has been sought to improve the selectivity and specificity of probe sets employed in detection and specific targeting of nucleic acids. We have evaluated the influence of "locked nucleic acid" residues on hybridization thermodynamics, counterions and hydration of DNA.RNA heteroduplex using spectroscopic and calorimetric techniques. One to three LNA substitutions have been introduced either at the adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) residues of the DNA strand. A complete thermodynamic profile for heteroduplex formation suggested that LNA-induced stabilization results from a favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, DeltaCp, accompanying the heteroduplex formation. Isothermal titration calorimetry measurements indicated an increase in binding affinity of the two strands as the LNA content of the heteroduplex is increased. Overall our result demonstrated that the effect of LNA-substitution at the thymine residue is more pronounced compared to the adenine residue. Furthermore, optical melting studies showed that, compared to an unmodified duplex, the formation of LNA-modified duplex is accompanied by a higher uptake of counterions and a lower uptake of water molecules. Our result, thus, presents a preliminary attempt toward the characterization of hybridization thermodynamics of the LNA-based probe-target sets, which will in turn aid in the selection of optimal conditions for hybridization experiments, and evaluation of the minimum probe-length required for hybridization and cloning experiments.
Biochemistry 02/2008; 47(4):1218-27. · 3.42 Impact Factor
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ABSTRACT: The LNA dinucleotide mimic of TpT whose two-sugar puckers are locked in the C3'-endo conformation selectively produces the corresponding cyclobutane pyrimidine dimer under 254 nm irradiation. In the natural series (TpT) the sugar puckers are in a major C2'-endo sugar conformation and the (6-4) photoproduct is also produced. Consequently, this study demonstrates that the C2'-endo conformation of the sugar pucker is necessary for (6-4) photoproduct formation.
Journal of the American Chemical Society 02/2008; 130(1):30-1. · 9.91 Impact Factor
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ABSTRACT: Novel, efficient and selective biocatalytic acylation / deacylation strategies have been used for the greener synthesis of precursors of different bicyclonucleosides. Biocatalytic methodology has also been developed for the separation of pyrano- and furanonucleosides, which is otherwise almost impossible to achieve by usual chemical approach.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: A study on the thermal stability of duplexes formed between unlocked nucleic acid (UNA) modified DNA and RNA oligonucleotides and complementary DNA and RNA is presented. The acyclic UNA monomers are shown to induce a decrease in duplex thermal stability.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: A synthetic route towards novel nucleosides with 1,4- dioxane as the sugar moiety has been developed. The dioxane moiety features a second anomeric center, which has been phosphitylated giving a diastereomeric mixture of the corresponding phosphoramidites.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: Two novel bicyclic nucleotide monomers have been developed for use as DNA mimics. Melting temperature studies showed that these modifications decrease binding affinity towards complementary DNA and RNA.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: The effect of locked nucleic acid (LNA) modification on hybridization kinetics of DNA duplex formation has been studied at varying salt concentration (Na(+) and Mg(2+)) using surface-plasmon resonance. The study suggested that the increased stability of LNA containing duplexes mainly originates from the slower dissociation rates constants of the duplexes. An increase in salt concentration increased the binding affinity of the individual duplexes by raising their association rate constants. Monitoring the change in binding affinity with respect to salt concentration revealed that of LNA-associated enhancement in helical stability mainly results from the changes in the non-electrostatic interactions upon duplex formation.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: A Locked Nucleic Acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring that is known to exhibit enhanced hybridization affinity towards complementary DNA and RNA, however the underlying thermodynamic basis for this observation are poorly understood. We have evaluated the influence of LNA residues on hybridization thermodynamics, counterions and hydration of DNA*DNA and DNA*RNA heteroduplex using spectroscopic and calorimetric techniques. Thermodynamic analysis for duplex formation using UV and differential scanning calorimetry suggested that LNA-induced stabilization results from a large, favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. The heat capacity change, Delta C(p), accompanying the duplex formation, obtained through DSC, has also been reported and has been used to furnish thermodynamic parameters at 37 degrees C. Furthermore, it was observed that relative to the formation of unmodified duplex, the formation of LNA-modified duplexes was accompanied by a higher uptake of counterions and a lower uptake of water molecules.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: A homogenous fluorescence dual-probe assay containing 2'-N-(pyren-1-ylmethyl)-2'-amino-LNA (locked nucleic acid) building blocks has been developed for effective mismatch-sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2'-amino-LNA through a short linker, are positioned at the 3' and 5' ends of a dual-probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal-denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch-induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual-probe assay constitutes a simple and sensitive system for the detection of single-nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual-probe constructs.
ChemBioChem 01/2008; 8(18):2240-8. · 3.94 Impact Factor
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ABSTRACT: RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8-17 deoxyribozymes. Four 8-17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues ("nucleation"), which precedes disruption of the local mRNA structure and completion of the binding process.
Journal of Biological Chemistry 01/2008; 282(49):35510-8. · 4.77 Impact Factor
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ABSTRACT: RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity
of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but
the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the
reaction of LNA-containing 8–17 deoxyribozymes. Four 8–17 constructs were designed to target sequences within the E6 mRNA
from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme)
were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular
cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate
complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding
LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes
retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets.
These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded
targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained
by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues (“nucleation”), which
precedes disruption of the local mRNA structure and completion of the binding process.
Journal of Biological Chemistry 12/2007; 282(49):35510-35518. · 4.77 Impact Factor
