Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents
Table 2
Methods to identify accessible sites in target RNAs.
Method
Type
Properties
References
MFold
IS
Algorithm finds minimal free energy (MFE) structure and set of lower energy structures. Display as pictorial structures or output as a single-stranded frequency map vector (probability estimator).
Uses MFold, SFold, OligoWalk, and in-house processing model to predict net probability of access in a region and to rank order the outcomes based on several parameters.
Search combinatorial ODN library for those entries able to bind to target RNA on basis of RNaseH of RNA: DNA hybrid, followed by primer extension analysis. Gel-based and cumbersome.
AS ODN sequence overlapping arrays are tiled onto silicon surfaces. Labeled target RNA is bound under defined conditions. Target binding to regions of the array identifies accessible regions.
Rich combinatorial library of hhRz sequences was used to cleave target RNA. First strand cDNA primed by Oligo-dT was followed by 3′ dG tailing, followed by PCR with a downstream gene-specific primerv and a poly-dC allowed amplification and sequencing to determine cleavage sites.
Uses probe for reverse transcription that has 3′ randomized region to screen for accessibility and constant region for PCR. Gene-specific upstream primers allow agarose gel-based mapping of accessible sites for antisense or ribozymes. Requires concurrent sequencing analysis for mapping.
Cleavage by AS or Rzs is followed by RT, 3′cDNA tailing, and then PCR using a tail-specific primer and a downstream gene-specific primer. Very sensitive.
Uses probe for reverse transcription that has 3′antisense to all NUH hhRz cleavage sites, followed by randomized region to screen for accessibility and 5′ constant region for PCR. Gene-specific upstream primers allow agarose gel-based mapping of accessible hhRz cleavage sites and their relative accessibility.
ODN with upstream and downstream constant regions embracing region of randomized sequence. Constant regions clamped by annealing complements. ssDNA region of MAST tags probes RNA target attached to beads. Annealing followed by washing, probe displacement, PCR, and sequencing. Little capacity to discriminate signal from noise.
Refined version of MAST in which the library is gene-specific or sequence-specific MAST tags against a target RNA are evaluated in competitive hybridization assay.
