Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, I.

doi:10.1093/nar/gkg801

Cited by 34 publications

(26 citation statements)

References 39 publications

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“…Other advantages of LNA include its close structural resemblance to native nucleic acids, which leads to very good solubility in physiological conditions and easy handling. In addition, owing to its charged phosphate backbone, LNA is nontoxic and can be delivered into cells using standard protocols that employ cationic lipid (28). All these properties are highly advantageous for a molecular tool for diagnostic applications.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

Yang

Lin

et al. 2007

Nucleic Acids Research

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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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Section: Introductionmentioning

confidence: 99%

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

Yang

Lin

et al. 2007

Nucleic Acids Research

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“…Catchpole and colleagues demonstrated that LNAs could invade plasmid DNA and remain associated with plasmid after delivery into cells (18). Giovannageli and colleagues showed that triplex-forming LNAs could block gene expression from plasmids inside permeabilized cells and bind to genomic DNA in intact cells (19,20).…”

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Inhibiting Gene Expression with Locked Nucleic Acids (LNAs) That Target Chromosomal DNA

et al. 2007

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Oligonucleotides containing locked nucleic acid bases (LNAs) have increased affinity for complementary DNA sequences. We hypothesized that enhanced affinity might allow LNAs to recognize chromosomal DNA inside human cells and inhibit gene expression. To test this hypothesis, we synthesized antigene LNAs (agLNAs) complementary to sequences within the promoters of progesterone receptor (PR) and androgen receptor (AR). We observed inhibition of AR and PR expression by agLNAs but not by analogous oligomers containing 2'-methoxyethyl bases or noncomplementary LNAs. Inhibition was dose dependent and exhibited IC 50 values of <10 nM. Efficient inhibition depended on the length of the agLNA, the location of LNA bases, the number of LNA substitutions, and the location of the target sequence within the targeted promoter. LNAs targeting sequences at or near transcription start sites yielded better inhibition than LNAs targeting transcription factor binding sites or an inverted repeat. These results demonstrate that agLNAs can recognize chromosomal target sequences and efficiently block gene expression. agLNAs could be used, for gene silencing, as cellular probes for chromosome structure, and therapeutic applications.The development of oligonucleotides and oligonucleotide mimics for sequence-specific recognition of chromosomal DNA inside cells confronts several challenges (1). Compounds must be able to enter cells, pass into the nucleus, and bind chromosomal DNA with high specificity. Binding must occur in spite of base-pairing at the target site and complexation of the genomic sequence with histones, transcription factors, and other DNA binding proteins. Once bound, the association of an oligomer with the chromosome must be sufficiently stable and long-lasting to affect gene expression. These challenges make recognition of DNA more complex than recognition of mRNA and overcoming them requires understanding the chemical, biophysical, and biological properties of native nucleic acids and their chemically modified analogs and mimics.We have shown that peptide nucleic acids 1 (PNAs) (Figure 1), a class of DNA/RNA mimic with an uncharged amide backbone (2,3), can target chromosomal DNA at transcription start sites and inhibit gene expression inside cells (4,5). Inhibition of gene expression by promotertargeted antigene PNAs (agPNAs, we use the term antigene to differentiate molecules that are complementary to chromosomal DNA from antisense oligomers that are complementary to mRNA) demonstrated that synthetic molecules could access sequence information at transcription start sites and that binding was sufficient to block expression.To whom correspondence should be addressed. Email: david.corey@utsouthwestern The PNAs used in our previous studies were mixed sequences (i.e. containing all four PNA bases) and were designed to recognize their targets by Watson-Crick base-pairing and strand invasion rather than by triple helix formation (4,5). With their neutral amide backbone and propensity to invade duplex DNA, PNAs have u...

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“…A study on a 15-mer pyrimidine TFO/LNA provided evidence that LNA-induced triplex stabilization is associated with a slower dissociation rate constant and a less unfavorable entropic contribution compared with the non-modified TFO (9). In previous works, TFO/LNAs have been used only for in vitro assays, including plasmid functionalization (10). However, LNAmodified oligonucleotides are appealing molecules: they have been successfully used as efficient antisense agents, even in vivo (for examples, see Refs.…”

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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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Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

Cited by 34 publications

References 39 publications

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons

Inhibiting Gene Expression with Locked Nucleic Acids (LNAs) That Target Chromosomal DNA

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

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