Homopurine-homopyrimidine sequences that flank certain actively transcribed genes are hypersensitive to single strand-specific nucleases such as S1. This has raised the possibility that an unusual structure exists in these regions that might be involved in recognition or regulation. Several of these sequences, including d(C-T) _n ⋅d(A-G) _n , are known to undergo a transition in plasmids to an underwound state that is hypersensitive to single strand-specific nucleases; this transition occurs under conditions of moderately acid p H and negative supercoiling. Chemical probes were used to examine the reactivity of a restriction fragment from a human U1 gene containing the sequence d(C-T) ₁₈ ⋅d(A-G) ₁₈ as a function of supercoiling and p H, and thus analyze the structure in this region. Hyperreactivity was seen in the center and at one end of the (C-T) _n tract, and continuously from the center to the same end of the (A-G) _n tract, in the presence of supercoiling and p H ≤ 6.0. These results provide strong support for a triple-helical model recently proposed for these sequences and are inconsistent with other proposed structures.

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21°C), nor shorter incubations at high temperatures (up to 95°C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37°C. However, incubation in fetal bovine or human sera at 37°C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

A vexing difficulty of the RNA world hypothesis is how RNA molecules of significant complexity could ever have evolved given their susceptibility to degradation. One way degradation might have been reduced is through low temperature. Here we report that truncated and fragmented derivatives of the hairpin ribozyme can catalyze ligation of a wide variety of RNA molecules to a given sequence in frozen solution despite having little or no activity under standard solution conditions. These results suggest that complex RNAs could have evolved in freezing environments on the early earth and perhaps elsewhere.

Small hairpin RNAs (shRNAs) are widely used in RNAi studies and typically consist of a stem of 19–29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3′ end. Compared with shRNAs with 21–29 bp stems, we have found that shRNAs with 19-bp or shorter stems (sshRNAs) possess some unique structure–activity features that depend on whether the antisense strand is positioned 5′ or 3′ to the loop (L- or R-type sshRNAs, respectively). L sshRNAs can have IC₅₀s in the very low picomolar range, and sshRNAs with nominal loop sizes of 1 or 4 nt were at least as active as those with longer loops. L sshRNAs remained highly potent even when the 3′ end of the antisense strand was directly linked with the 5′ end of the sense strand. In this case, the sense strand can be shorter than the antisense strand, and the loop can be formed entirely by the 3′ end of the antisense strand. Monomer sshRNAs are not processed by recombinant Dicers in vitro. Although they can form dimers that are sometimes Dicer substrates, their RNAi activity is not dependent on the formation of such structures. Our findings have implications for the mechanism of action of sshRNAs, and the ability to design highly potent shRNAs with minimal length is encouraging for the prospects of the therapeutic use of direct-delivered shRNAs.

Samples of DNA which contain adducts of a new psoralen derivative, but no cross-links, have been prepared by irradiating mixtures of DNA and the derivative with single, 15-nanosecond pulses of laser light. Succeeding pulses introduce cross-links. The ability to rapidly and selectively create monoadducts and corss-links may allow the use of psoralens as probes of dynamic processes in living cells.