LNA and α-L-LNA: Towards Therapeutic Applications

Wengel, Jesper; Vester, Birte; Lundberg, L.; Douthwaite, Stephen; Sørensen, Mads D.; Babu, B. Ravindra; Gait, Michael J.; Arzumanov, Andrey A.; Petersen, Michael; Nielsen, Jakob T.

doi:10.1081/ncn-120021963

Cited by 24 publications

(7 citation statements)

References 4 publications

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“…Stability of synthetic oligonucleotides in diverse biological media, including human serum (HS), is of crucial significance for their application as diagnostic and therapeutic tools in vivo. 45 Chemical modification with synthetic nucleotide analogues, especially LNA, 46 is known to dramatically improve the enzymatic stability of oligonucleotides. 42,46 In our study we investigated the stability of the novel POCs in a weakly diluted HS, a complex medium containing several classes of enzymes, which is very similar to the media of in vivo studies and clinical trials.…”

Section: Stability Of Pocs In Human Serummentioning

confidence: 99%

Peptide–LNA oligonucleotide conjugates

et al. 2013

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Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly improve biomolecular recognition by synthetic nucleic acid analogues. Circular dichroism (CD) measurements showed no distortion of the duplex structure by the incorporated peptide chains while studies in human serum indicated superior stability of the POCs compared to LNA/DNA mixmers and unmodified DNA references. Molecular modeling suggests strong interactions between positively charged regions of the peptides and the negative oligonucleotide backbones which leads to clamping of the peptides in a fixed orientation along the duplexes.

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Section: Stability Of Pocs In Human Serummentioning

confidence: 99%

Peptide–LNA oligonucleotide conjugates

et al. 2013

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“…ribonucleotides with a 2Ј-O,4Ј-C-methylene linkage that effects conformational fixation of the furanose ring in a C3Ј-endo conformation (3,4). LNA-containing oligonucleotides have been recently shown to enhance triplex stability and to alleviate in part the sequence constraints imposed by the triple helical recognition motifs (5,6). Only a few hybridization properties of LNA-modified TFOs (TFO/LNAs) have been reported so far, and they all concern (T,C)-containing TFO/LNAs.…”

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confidence: 99%

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

Brunet

Alberti

Perrouault

et al. 2005

Journal of Biological Chemistry

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Triplex-forming oligonucleotides (TFOs), as DNAbinding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine⅐oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect.The design of synthetic non-protein molecules able to control DNA-associated biological functions through their interaction with a specific DNA sequence represents a very attractive approach. Among DNA code-reading molecules, triplex-forming oligonucleotides (TFOs) 1 are able to bind to the major groove of oligopyrimidine⅐oligopurine regions in doublestranded DNA. A series of results have validated triplex-based approaches at the molecular and cellular levels: triplexes have been shown to interfere with transcription (initiation and elongation), replication, repair, and recombination (1, 2). However, the intracellular efficiency of TFOs still has to be improved.One possible approach consists of increasing the stability of the non-covalent triple helices under physiological conditions to reach binding affinities comparable to those of DNA-associated regulatory proteins. To this end, a variety of chemically modified nucleic acids have been developed. Among them are locked nucleic acids (LNAs) that contain LNA nucleotide monomers, i.e. ribonucleotides with a 2Ј-O,4Ј-C-methylene linkage that effects conformational fixation of the furanose ring in a C3Ј-endo conformation (3, 4). LNA-containing oligonucleotides have been recently shown to enhance triplex stability and to alleviate in part the sequence constraints imposed by the triple helical recognition motifs (5, 6). Only a few hybridization properties of LNA-modified TFOs (TFO/LNAs) have been reported so far, and they all concern (T,C)-containing TFO/LNAs. It has been shown that fully modified TFO/LNAs failed to bind to double-stranded DNA (7, 8), likely due to conformational restraint of TFO/LNA. However, alternating DNA and LNA nucleotides in TFO sequences is appropri...

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“…To achieve this, we prepared a series of (Ϫ) DNA templates containing locked nucleic acid (LNA) analogs (47,48). These 2Ј-O-4Ј-C-methylene-linked bicyclic analogs (Fig.…”

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Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/ DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (؊) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/ DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA⅐rU tract immediately 5 of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT⅐rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC⅐rG and neighboring dT⅐rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3 -end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (؉5/؉6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 ؉ 5/؉6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.Minus (Ϫ)-strand DNA synthesis in retroviruses is accompanied by degradation of viral RNA of the RNA/DNA replication intermediate. These are events mediated by the N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) 1 catalytic centers of the multifunctional reverse transcriptase (RT), respectively (1). Correct positioning of nucleic acids to facilitate each of these steps clearly requires its specific interaction with structural motifs of the virus-coded polymerase. Crystallographic (2-4) and biochemical analysis (5-13) of human immunodeficiency virus type 1 (HIV-1) RT identified the primer grip of the p66 thumb as a motif critical for positioning the primer 3ЈOH before nucleophilic attack on the incoming dNTP. Immediately adjacent to this motif, the minor groove binding track (MGBT) or translocation track is proposed to mediate translocation of the polymerization machinery and act as a sensor of nucleic acid geometry (14 -19). Recently, it was proposed that the RNase H primer grip (RHPG), involving portions of the p66 connection subdomain and RNase H domain, interacts with the DNA strand of an RNA/DNA hybrid, providing the RNA with a trajectory appropriate for hydrolysis within the RNase H catalytic center (4, 20, 21). These combined activities effectively prepare nascent (Ϫ) DNA for use as template for plus (ϩ)-strand, DNA-dependent DNA synthesis.A critical...

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LNA and α-L-LNA: Towards Therapeutic Applications

Cited by 24 publications

References 4 publications

Peptide–LNA oligonucleotide conjugates

Peptide–LNA oligonucleotide conjugates

Exploring Cellular Activity of Locked Nucleic Acid-modified Triplex-forming Oligonucleotides and Defining Its Molecular Basis

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

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