Discovering Antisense Reagents by Hybridization of RNA to Oligonucleotide Arrays

Southern, Edwin M.; Milner, Natalie; Mir, Kalim U.

doi:10.1002/9780470515396.ch4

Cited by 11 publications

(4 citation statements)

References 11 publications

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“…So, we have no correlation between in vitro accessibility and in vivo efficacy, and this is a limitation for our study. Several studies have a finding of a significant correlation between the degree of translation inhibition in cell-free systems and predicted that hybridization potential is consistent with results from in vitro and in vivo studies of antisense reagents activities [16,58,59,64]. Conseqently, to verify the activity of Dz1751 in vivo will be the next work for us.…”

Section: Discussionmentioning

confidence: 54%

Ezrin mRNA target site selection for DNAzymes using secondary structure and hybridization thermodynamics

et al. 2011

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Ezrin, a membrane organizer and linker between plasma membrane and cytoskeleton, is well documented to play an important role in the metastatic capacity of cancer cells especially for osteosarcoma cells. It has provided an ideal target for cancer gene therapy. RNA-cleaving 10-23 DNAzymes, consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, can cleave the target mRNA at purine-pyrimidine dinucleotide effectively. In the present study, we designed and screened the target sites for 10-23 DNAzymes against ezrin mRNA by using multiple computational methods with combination of secondary structural and hybridization thermodynamic parameters. Then, we testified the activities of the DNAzymes directed against these selected target sites in vitro. Our results show that AU1751 is the most effective target site of ezrin mRNA for DNAzymes because of its ideal secondary structure and hybridization thermodynamics. So, there is a significant correlation between the multiple computational methods and the efficacy of the corresponding DNAzymes. These provide a rational, efficient way for DNAzymes selection.

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Section: Discussionmentioning

confidence: 54%

Ezrin mRNA target site selection for DNAzymes using secondary structure and hybridization thermodynamics

et al. 2011

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“…While it is not firmly established that susceptibility to RNase-H-mediated cleavage and/or binding of oligonucleotides indicates that such sites are necessarily accessible in vivo [Branch, 1998], a variety of studies support this contention [Milner et al, 1997; Southern et al, 1997; Duan et al, 2006; Kierzek et al, 2006]. In many examples, accessible sites identified in vitro have been found to also be accessible in vivo [Milner et al, 1997; Southern et al, 1997; Pan et al, 2001]. Nevertheless, failure of the miRs aligning within the 3′-UTR of KRT, or within the R9 region, to inhibit translation could be due to protein masking of the sites in vivo.…”

Section: Resultsmentioning

confidence: 99%

MicroRNAs align with accessible sites in target mRNAs

Pan¹,

Xin²,

Clawson³

2009

J of Cellular Biochemistry

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The importance of microRNAs (miRs) in control of gene expression is now clearly recognized. While individual microRNAs are thought to target hundreds of disparate mRNAs via imperfect base pairing, little is known about the characteristics of miR target sites. Here we show that the miRs can be aligned with empirically identified accessible sites in a target RNA (Cytokeratin 19, KRT), and that some of the aligned miRs functionally down-regulate KRT expression post-transcriptionally. We employed an RNase-H-based random library selection protocol to identify accessible sites in KRT RNA. We then aligned the Sanger Institute database collection of human miRs to KRT mRNA, and also aligned them using the web-based MicroInspector program. Most miRs aligned with the accessible sites identified empirically; those not aligned with the empirically identified sites also functioned effectively in RNase-H-based assays. Similar results were obtained with a second target RNA (Mammoglobin). Transient transfection assays established that some of the miRs which aligned with KRT significantly down-regulated it at the protein level, with no effect on RNA level. The functionally effective miRs aligned within the coding region of KRT, whereas a number of miRs which aligned with the 3′-untranslated region did not produce down-regulation.

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“…They are used for the analysis and comparison of gene expression levels (1–3), resequencing genes to identify mutations (4–6), analysis of sequence polymorphism on a large scale (7,8), optimization of antisense oligonucleotides (9), basic studies of molecular hybridization (10,11) and analysing DNA–protein interactions (12). Several methods have been developed to fabricate arrays.…”

Section: Introductionmentioning

confidence: 99%

Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication

Egeland

Southern²

2005

Nucleic Acids Research

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We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites. The acid is produced by electrochemical oxidation controlled by an array of independent microelectrodes. Deblocking is complete in a few seconds, when competing side-product reactions are minimal. We demonstrate the successful synthesis of 17mers and discrimination of single base pair mismatched hybrids. Features generated in this study are 40 μm wide, with sharply defined edges. The synthetic technique may be applicable to fabrication of other molecular arrays.

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Discovering Antisense Reagents by Hybridization of RNA to Oligonucleotide Arrays

Cited by 11 publications

References 11 publications

Ezrin mRNA target site selection for DNAzymes using secondary structure and hybridization thermodynamics

Ezrin mRNA target site selection for DNAzymes using secondary structure and hybridization thermodynamics

MicroRNAs align with accessible sites in target mRNAs

Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication

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