Rational selection of antisense oligonucleotide sequences

Smith, Lise; Andersen, Klaus B.; Hovgaard, Lars; Jaroszewski, Jerzy W.

doi:10.1016/s0928-0987(00)00100-7

Cited by 43 publications

(26 citation statements)

References 26 publications

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“…For instance, a short sequence of bases (as short as 13 -25 bases is specific; refs. 12, 13) can be used to specifically hybridize to mRNA in areas for ribosomal binding (12 -14), which are usually the 5V untranslated region or the AUG start codon (14). This prevents ribosomal binding and translation of the mRNA.…”

Section: Introductionmentioning

confidence: 99%

“…The advantage of this technique is that after cleavage, the ASODN is free to associate with another strand of mRNA allowing one ASODN to degrade several mRNA strands. Any sequence within the mRNA can be used as a target for the ASODN, giving it a variety of areas to target (14). ASODN can also be used to target the capping and polyadenylations sites of mRNA, which destabilizes it.…”

Section: Introductionmentioning

confidence: 99%

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Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer

Cao¹,

Shinohara²,

Niermann³

et al. 2005

Molecular Cancer Therapeutics

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Murine double minute 2 (MDM2) inhibits p53-mediated functions, which are essential for therapies using DNAdamaging agents. The purpose of this study was to determine whether MDM2 inhibition enhances the radiosensitivity of a lung cancer model. The effects of MDM2 inhibition on tumor vasculature were also studied. Transient transfection of H460 lung cancer cells and human umbilical vascular endothelial cells (HUVEC) with antisense oligonucleotides (ASODN) against MDM2 resulted in a reduced level of MDM2 and increased levels of p21 and p53. Clonogenic assays showed that inhibition of MDM2 greatly decreased cell survival following irradiation. Quantification of apoptotic cells by 7-aminoactinomycin D staining and of senescent cells by X-gal staining showed that both processes were significantly increased in H460 cells treated with MDM2-specific ASODN and radiation. H460 xenografts that were treated with MDM2 ASODN plus radiotherapy also showed significant growth delay (P < 0.001) and increased apoptosis by terminal deoxynucleotidyl transferase -mediated nick end labeling staining. HUVECs transfected with MDM2-specific ASODN showed impaired viability and migration with decreased tube formation. Doppler studies showed that tumor blood flow was compromised when H460 xenografts were treated with MDM2-specific ASODN and radiation. A combination of radiotherapy and inhibition of MDM2 through the antisense approach results in improved tumor control in the H460 lung cancer model. This implies that a similar strategy should be investigated among patients with locally advanced lung cancer, receiving thoracic radiotherapy. [Mol Cancer Ther 2005;4(8):1137 -45]

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer

Cao¹,

Shinohara²,

Niermann³

et al. 2005

Molecular Cancer Therapeutics

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“…Identification of optimal antisense inhibitors has traditionally been a laborious empirical process, requiring individual testing of constructs. Prediction of accessible sites by thermodynamic modeling, chemical probing, and/or enzymatic mapping of RNA secondary structure has permitted a more rational and modestly reliable approach to antisense design (46,57). Recently, combinatorial methods with sequence-randomized oligonucleotide libraries have been used to exhaustively screen all target sites within a given RNA.…”

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confidence: 99%

Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Smith

2004

Journal of Viral Hepatitis

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The 3 -terminal sequences of hepatitis C virus (HCV) positive-and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3 untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3 terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3 -terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positivestrand 3 -X region RNA in vitro.The untranslated regions (UTRs) of the hepatitis C virus (HCV) positive-strand genome and negative-strand intermediate contain cis-acting sequences essential for viral translation and RNA replication. The 341-nucleotide (nt) 5Ј-UTR of the positive strand acts as an internal ribosome entry site (IRES) to direct cap-independent translation of the single ϳ3,000-codon-long HCV open reading frame (38, 40). The tripartite 3Ј-UTR consists of a short upstream variable region, a central poly(U)-polypyrimidine stretch of variable length, and a terminal highly conserved 98-nt sequence (38). With the exception of the variable region, all sequence elements in the 3Ј-UTR are necessary for intracellular replication of HCV RNA (16,29,56).A number of in vitro studies (26,35,36,39,58) have established that the terminal 98-nt X region sequence can serve as a minimal template for de novo initiation of negative-strand synthesis by the viral RNA-dependent RNA polymerase, NS5B. The 3Ј terminus of the HCV negative strand reportedly plays an analogous role in positive-strand RNA synthesis (25,36,39). The binding of HCV 3Ј termini to various host proteins may exert subtle effects on IRES-mediated translation (16,23,34,55) or protect viral transcripts from degradation by cytoplasmic RNases (15,48,49).The replicative and protein-binding functions of heteropolymeric regions in the HCV 3Ј termini are, in many instances, dependent on the ability of the primary sequence to fold into higher-order RNA structure. In vitro, the X region sequence is capable of adopting a three-stem-loop structure, with the 3Ј-terminal 46 nt forming a thermodynamically stable stem-loop, SL I (6). Mutational analysis indicates that the duplex structure forming the base of SL I influences both the site and efficiency of de novo initiation by NS5B (26,35). Preservation of the interior stem-...

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“…To take advantage of antisense technologies, antisense libraries have been constructed using conventional ASoligos (24). However, selection of an effective target site for each gene is time-consuming and often inconclusive (16,17). It is generally accepted that construction of an extensive AS-oligo library able to target most human genes would take years to finish, necessitating a way to overcome these problems.…”

Section: Discussionmentioning

confidence: 99%

Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA

2004

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Rational selection of antisense oligonucleotide sequences

Cited by 43 publications

References 26 publications

Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer

Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer

Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA

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