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J. M. Sullivan, R. Taggart; Novel and Enhanced Approaches to Determine Local mRNA Accessibility. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4605.
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© ARVO (1962-2015); The Authors (2016-present)
Accessible regions in Rho mRNA containing NUH↓ cleavage sites were predicted using a battery of computational tools including MFold, SFold, OligoWalk and in-house algorithms. gsMAST tested accessibility at 22 predicted accessible and inaccessible regions by competitive solution hybridization of individual oligonucleotides complementary to discrete 18 nt regions spanning hhRz cleavage sites. Target was biotin-labeled full-length (1532 nt) Rho mRNA. After extensive washing, target mRNA bound probes were eluted from streptavidin coated mag-beads, PCR amplified, cloned, and isolates were DNA sequenced. Control sense probe was included in all screens. Relative accessibility was determined by the recovery frequency of antisense sequences corresponding to discretely probed regions, and statistically evaluated (Χ2). cMARS was performed by 1st strand cDNA synthesis on mRNA primed with 4 oligonucleotide libraries (one for each NUH↓ site class, e.g. GUH↓) each terminated at the 3’ end with antisense sequence (5’-DAN-3’) to a hhRz cleavage site (5-NUH-3’), followed upstream by a random six-mer, and then a constant amplifiable sequence. Sites accessible for cDNA synthesis initiation were mapped by PCR with a series of opsin specific (upstream) primers. Products were screened for size and abundance by gel electrophoresis, and then cloned for DNA sequencing.
9 of 22 gsMAST probes exhibited hybridization and 6 of these sites embraced over 90% of the clones sampled (Χ2 analysis, p ≤ 0.001). gsMAST confirmed accessibility and inaccessibility predicted by the in silico platform. Control sense probes were never isolated. cMARS identified 12 regions containing hhRz cleavage sites, including several revealed by computational and gsMAST approaches. Accessible sites support hhRz knockdown.
mRNA accessible site mapping has reduced the scope of our hhRz testing 1-log order. These approaches are strongly validated for Rho mRNA, which contains over a hundred and fifty mutations that cause various forms of retinal degeneration, and is a target for a mutation-independent hhRz therapy. These tools will support mapping of other disease mRNAs for therapy development.
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