Figure 1

Hairpin anatomy and the most common stem lengths. (A) Typical hairpin design begins with an optimal siRNA core that is extended in one or both directions to a total stem length of 19 to 29 bp. The 3' end of the upper siRNA strand is connected to the 5' end of the lower siRNA strand by a loop sequence. The shRNA stem length is defined as the stretch of sequence between the terminally paired nucleotides. The upper strand of the stem is the 'sense' strand which is designed to give rise to the siRNA 'passenger' strand. The lower strand is the 'anti-sense' strand and is designed to give rise to the siRNA 'guide' strand. (*) The stem region towards the free end of the hairpin is referred to as the base terminus whereas the stem region towards to the loop end is referred to as the loop terminus. The point at which the stem meets the loop is referred to as the stem-loop junction. (B) A commonly used loop sequence (UUCAAGAGA) (used in 60% of surveyed studies) is predicted to internally pair (UU.. to ..GA) resulting in an unintended shift in the stem-loop junction. (C) 101 studies employing expressed shRNA were surveyed and each hairpin was scored for stem length. The stem lengths were found to range from 19 - 29 bp, with the most commonly designed stem length being 19 bp (58% of all hairpins). When designed stem lengths are adjusted for additional loop sequence the most common length is 21 bp (60%).