Short hairpin RNA loop design for the facilitation of sequence verification.

The suppression of protein expression using RNA interference (RNAi) has become a powerful tool for the study of gene function (1). Initial applications of RNAi in mammalian cells utilized 21-bp RNA duplexes referred to as small interfering RNAs (siRNAs) (2). Although highly effective, siRNAs were found to be relatively short lived and therefore suboptimal for studies requiring long-term gene suppression. To overcome this limitation, vector expression systems for the DNA directed transcription of single-stranded RNA molecules containing forward and reverse complements separated by a loop segment were devised (3–5). Following transcription, the loop segment enables the formation of a short hairpin RNA (shRNA) structure that can be recognized and processed into siRNAs by the RNase III family nuclease, Dicer (6). Dicer-derived siRNAs can, in turn, be more effective than chemically synthesized siRNAs in inducing messenger RNA (mRNA) degradation (7). A particularly useful aspect of RNAi is the ability to differentially suppress genes or variations of genes that differ by even a single nucleotide. Conversely, an unintended single nucleotide mismatch between an shRNA and its target sequence may be sufficient to abrogate gene suppression (8). It is imperative therefore for shRNA encoding plasmids to be sequence-verified prior to use. The use of standard sequencing methods using DNA polymerases has, however, been problematic because of the inherent secondary structure of the shRNA encoding region. Additives such as dimethyl sulfoxide (DMSO) and betaine, as well as a series of commercially available proprietary compounds, have been used in a trial-and-error fashion to relax the hairpin secondary structure of the single-stranded DNA plasmids during sequencing reactions. We sought to design a universal loop region that could facilitate the sequence verification of shRNA encoding plasmids. While several algorithms have been developed for the design of the stem portion of an shRNA molecule, such systematic studies have not been reported for the loop region. Given the wide variety of loop region sequences used by individual investigators and the ability of Dicer to efficiently process RNA duplexes lacking loop regions (9), we inferred that the actual nucleotide sequence of the loop is likely subject to minimal constraints. We therefore chose a loop sequence beginning with an " A " followed by the recognition site for the nonpalindromic restriction endonuclease BmgBI (5′-A[CACGTC]-3′). Due to its nonpalindromic nature, there is no contribution of this sequence to the stem portion of the shRNA molecule. Likewise, there are no restrictions on the …