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.

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here,weshowthat incorporation of LNAsubstantially enhances serum half-life of siRNA’s, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA’s promise in converting siRNA from a functional genomics technology to a therapeutic platform.

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificityonall possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.

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.

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.