Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statistical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5'-anti-sense (AS) terminal base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal stability (Delta0.5 kcal/mol) at the 5'-AS end than nonfunctional duplexes. Average internal stability of siRNA molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as “aptamers”, are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Amgen, Inc. One Amgen Center Drive nucleoprotein complex that includes at least two members of the RISC, further linking siRNA and miRNA pro-Thousand Oaks, California 91320 2 Dharmacon, Inc. cessing (Mourelatos et al., 2002). Unlike siRNAs, which regulate mRNA levels through a cleavage event, miRNAs 1376 Miners Drive, #101 Lafayette, Colorado 80026 function by attenuating translation (Hutvá gner and Zamore, 2002). However, it was recently reported that a well-characterized let-7 miRNA, which naturally directs translational attenuation, can also cleave mRNA so long Summary as it is composed of a sequence that is completely complementary to the target (Hutvá gner and Zamore, Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer 2002). The finding that an miRNA can function as an siRNA (Doench et al., 2003; Zeng et al., 2003) indicates and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statis-that the degree of sequence identity between the regulatory RNA and its target may be the key factor in de-tical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor termining which form of posttranscriptional gene silencing is exploited. hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5-anti-sense (AS) terminal Several studies have shown that duplex unwinding is critical for the processing of both dsRNA and pre-miRNA base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal hairpin precursors (Bernstein et al., 2001; Nicholson and Nicholson, 2002) and is necessary for the formation of stability (⌬0.5 kcal/mol) at the 5-AS end than nonfunctional duplexes. Average internal stability of siRNA silencing-competent (activated) RISC (RISC*), thus highlighting the importance of helicase activity in the RNAi molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic process (Nykanen et al., 2001). In addition, recent studies in D. melanogaster cell lysates suggest that sequence thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA asymmetry and strand instability in the pre-miRNA hairpin precursor or siRNA duplex may contribute to selec-play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved tive strand processing and entry into RISC* (Schwarz et al., 2003 [this issue of Cell]). Taken together, these stud-in duplex unwinding and strand retention by RISC. ies hint that a correlation may exist between the thermodynamic stability of siRNAs and miRNAs and their ability

The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.