Application of siRNA in high-throughput fashion is still in its early phase although the principle has been established for three years. In this review, we outline the different vector-based siRNA delivery platforms as well as resources that are becoming available for high-throughput applications, and some initial outcomes of vector siRNA high-throughput screening efforts using vector encoded siRNA. It is expected that further improvement of the siRNA technology and availability of the siRNA resources will help to materialize the potential of siRNA for functional genomics and drug target validation.

Only a small fraction of all siRNAs are effective in silencing their target genes, and siRNA efficacy can only be determined experimentally. Previously described reporter-based siRNA validation methods all rely on the availability of physical cDNA clones, and this limits the high throughput applicability of the method. In the current report, we used short synthetic DNA fragment containing a siRNA targeting site, instead of cDNA, to fuse with a reporter gene. When targeting such transcripts with different siRNAs, we found that such constructs can faithfully report the efficacy of the corresponding siRNAs in a sequence specific manner, even when the inserted DNA fragment is essentially only long enough to cover the targeting site. The efficacy of both vector-based siRNA and synthetic siRNA can be evaluated using this system. Since only readily available short synthetic DNA fragments are needed for forming the evaluation vector, this method provides an appealing way of validating siRNAs in high throughput.

We have developed a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products on unmodified glass surfaces. By covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solutions we have generated a new class of modified nucleic acids, namely silanized nucleic acids. Such silanized molecules can be immobilized instantly onto glass surfaces after manual or automated deposition. This method provides a simple and rapid, yet very efficient, solution to the immobilization of prefabricated oligonucleotides and DNA for chip production.

A solution-based method, mRNA accessible site tagging (MAST), has been developed to map the accessible sites of any given mRNA in high throughput fashion. mRNA molecules were immobilized and hybridized to randomized oligonucleotide libraries. Oligonucleotides speci®cally hybridized to the mRNA were sequenced and found to be able to precisely define the accessible sites of the mRNA. A number of ways were used to validate the accessible sites defined by the MAST process. Mapping of rabbit b-globin mRNA demonstrates the efficacy and advantage of MAST over other technologies in identifying accessible sites. Antisense oligonucleotides designed according to the accessible site map of human RhoA and Renilla luciferase mRNA result in knockdown effects that are in good correlation with the degrees of accessibility. The MAST methodology can be applied to mRNA of any length using a universal protocol.