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cially 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

Short hairpin RNA loop design for the facilitation of sequence verification