Metal ions: supporting actors in the playbook of small ribozymes.

Since the 1980s, several small RNA motifs capable of chemical catalysis have been discovered. These small ribozymes, composed of between approximately 40 and 200 nucleotides, have been found to play vital roles in the replication of subviral and viral pathogens, as well as in gene regulation in prokaryotes, and have recently been discovered in noncoding eukaryotic RNAs. All of the known natural small ribozymes - the hairpin, hammerhead, hepatitis delta virus, Varkud satellite, and glmS ribozymes--catalyze the same self-cleavage reaction as RNase A, resulting in two products, one bearing a 2'-3' cyclic phosphate and the other a 5'-hydroxyl group. Although originally thought to be obligate metalloenzymes like the group I and II self-splicing introns, the small ribozymes are now known to support catalysis in a wide variety of cations that appear to be only indirectly involved in catalysis. Nevertheless, under physiologic conditions, metal ions are essential for the proper folding and function of the small ribozymes, the most effective of these being magnesium. Metal ions contribute to catalysis in the small ribozymes primarily by stabilizing the catalytically active conformation, but in some cases also by activating RNA functional groups for catalysis, directly participating in catalytic acid-base chemistry, and perhaps by neutralizing the developing negative charge of the transition state. Although interactions between the small ribozymes and cations are relatively nonspecific, ribozyme activity is quite sensitive to the types and concentrations of metal ions present in solution, suggesting a close evolutionary relationship between cellular metal ion homeostasis and cation requirements of catalytic RNAs, and perhaps RNA in general.

A bird's eye view tracking slow nanometer-scale movements of single molecular nano-assemblies.

Recent improvements in methods of single-particle fluorescence tracking have permitted detailed studies of molecular motion on the nanometer scale. In a quest to introduce these tools to the burgeoning field of DNA nanotechnology, we have exploited fluorescence imaging with one-nanometer accuracy (FIONA) and single-molecule high-resolution colocalization (SHREC) to monitor the diffusive behavior of synthetic molecular walkers, dubbed "spiders," at the single-molecule level. Here we discuss the imaging methods used, results from tracking individual spiders on pseudo-one-dimensional surfaces, and some of the unique experimental challenges presented by the low velocities (approximately 3 nm/min) of these nanowalkers. These experiments demonstrate the promise of fluorescent particle tracking as a tool for the detailed characterization of synthetic molecular nanosystems at the single-molecule level.