Enhanced Inhibition of Transcription Start by Targeting with 2′‐OMe Pentaribonucleotides Comprising Locked Nucleic Acids and Intercalating Nucleic Acids

Filichev, Vyacheslav V.; Vester, Birte; Hansen, Lykke Haastrup; Aal, Mohammed Taha Abdel; Babu, B. Ravindra; Wengel, Jesper; Pedersen, Erik B.

doi:10.1002/cbic.200400457

Cited by 12 publications

(13 citation statements)

References 16 publications

(4 reference statements)

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“…LNAs were designed to be complementary to sequences at the transcription start site and to regions further upstream or downstream. Pedersen and colleagues had previously shown that LNA pentanucleotides could block transcription by E. coli polymerase in cell free assays (26), a result similar to that achieved in classic studies by Sigman and coworkers using RNA pentanucleotides (27).…”

Section: Design Of Lnassupporting

confidence: 70%

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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Section: Design Of Lnassupporting

confidence: 70%

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

et al. 2007

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“…Duplexes with other interstrand zipper arrangements of X monomers only exhibit marginal signal intensity at 495 nm (results not shown). Pyrene emission signals at 495 nm are routinely attributed to pyrene-pyrene excimers41,42,45,51–55, suggesting that the pyrene moieties of the two X monomers comprising the energetic hotspots in Invader LNA probes are separated by ~3.4 Å56 (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Examples include minor groove binding polyamides21, TFOs capable of recognizing all four Watson-Crick base pairs via the major groove22,23, optimized PNAs24,25, antigene (ag) PNA26,27, agLNA28,29, “Zorro” LNA30, pseudo-complementary (pc) DNA31,32, pcPNA33–38, and other approaches39–41. While these emerging probe strategies are promising, there remains a need for a probe technology that allows fast, efficient and specific dsDNA targeting at physiologically relevant ionic strengths with minimal sequence restrictions and uncomplicated “DNA-like” handling (adequate aqueous solubility, compatibility with delivery agents).…”

Section: Introductionmentioning

confidence: 99%

Invader LNA: Efficient targeting of short double stranded DNA

Kumar

Hrdlicka

2010

Org. Biomol. Chem.

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Despite progress with triplex-forming oligonucleotides or helix-invading peptide nucleic acids (PNAs), there remains a need for probes facilitating sequence-unrestricted targeting of double stranded DNA (dsDNA) at physiologically relevant conditions. Invader LNA probes, i.e., DNA duplexes with "+1 interstrand zipper arrangements" of intercalator-functionalized 2'-amino-α-L-LNA monomers, are demonstrated herein to recognize short mixed sequence dsDNA targets. This approach, like pseudo-complementary PNA (pcPNA), relies on relative differences in stability between probe duplexes and the corresponding probe:target duplexes for generation of a favourable thermodynamic gradient. Unlike pcPNA, Invader LNA probes take advantage of the "nearest neighbour exclusion principle", i.e., intercalating units of Invader LNA monomers are poorly accommodated in probe duplexes but extraordinarily well tolerated in probe-target duplexes (ΔT m /modification up to +11.5 °C). Recognition of isosequential dsDNA-targets occurs: a) at experimental temperatures much lower than the thermal denaturation temperatures (T m 's) of Invader LNAs or dsDNA-targets, b) at a wide range of ionic strengths, and c) with good mismatch discrimination. dsDNA recognition is monitored in real-time using inherent pyrene-pyrene excimer signals of Invader LNA probes, which provides insights into reaction kinetics and enables rational design of probes. These properties render Invader LNAs as promising probes for biomedical applications entailing sequence-unrestricted recognition of dsDNA.

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“…There is also a correlation between the thermal stability and the formation of an excimer band. Thus, T m values of duplexes showing an excimer band ( ON14 / ON22 ,52, 53 ON18 / ON26 , and ON20 / ON27 ) are higher than the T m values of duplexes with no excimer band in fluorescence spectra ( ON15 / ON23 , ON16 / ON24 , and ON17 / ON25 ). This means that proper interstrand overlapping/stacking of pyrene residues as a pseudo‐pair in the middle of the DNA duplex results in the formation of an excimer band.…”

Section: Resultsmentioning

confidence: 83%

“…We have recently observed that placing two INA monomers opposite each other in the duplex led to the formation of so‐called easily denaturing nucleic acids53, 54 in which each of the INA sequences has a preference in binding to the complementary unmodified DNA of the same length rather than forming a duplex with each other 26. 52, 53 A similar tendency was observed for o TINA with 1‐ethynylpyrene, that is, the duplex with an inserted pair of 5 ( ON18 / ON26 ) had lower T m (42.0 °C) than duplexes with the complementary DNA molecules ( ON18 / ON21 and ON26 / ON13 with T m of 46.5 and 44.5 °C, respectively). None of the duplexes with a bulged pair of intercalators in the middle of the sequence showed an increased thermal stability in comparison with the wild‐type duplex.…”

Section: Resultsmentioning

confidence: 99%

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

Filichev

Astakhova

Malakhov

et al. 2008

Chemistry A European J

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A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

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Enhanced Inhibition of Transcription Start by Targeting with 2′‐OMe Pentaribonucleotides Comprising Locked Nucleic Acids and Intercalating Nucleic Acids

Cited by 12 publications

References 16 publications

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

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

Invader LNA: Efficient targeting of short double stranded DNA

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

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