Review Focus on Function: Single Molecule RNA Enzymology Mark A. Ditzler, 1 Elvin A. Alema ´n, 2 David Rueda, 2 Nils G. Walter 3 1 Biophysics Research Division, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 48109 2 Department of Chemistry, Wayne State University, Detroit, MI 48202 3 Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 48109 Received 3 July 2007; revised 24 July 2007; accepted 24 July 2007 Published online 8 August 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20819 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION A detailed understanding of the biopolymer ribonu- cleic acid (RNA) is of great importance throughout the life sciences. RNA-coding genes are now recog- nized to be far more abundant in eukaryotes than their protein-coding counterparts and are essential to the central biochemical processes within all living cells. 1–3 RNA is responsible for the synthesis of all proteins within the cell, plays a central role in replication of many viruses, regu- lates gene expression in both bacteria and eukaryotes, is involved in the maintenance, processing, modification, and editing of genetic information, and probably carries out a host of still unknown cellular processes. The discovery of the cata- lytic capabilities of group I introns 4 and RNase P, 5 coupled with the knowledge that certain viral genomes are composed entirely of RNA, established RNA as unique in nature for its ability to both store genetic information and catalyze chemical reactions. The dual genetic and catalytic role of RNA lends tre- mendous support to the hypothesis that purely RNA-based life predated the emergence of both protein and DNA. 6–8 In addition to their important functions in nature, catalytic RNAs have been used to derive RNA-based therapeutics. 9,10 Our understanding of the molecular underpinnings of organ- isms, and possibly the origin of life, as well as the development of new medicines, therefore, significantly depend on our abil- ity to dissect the fundamental properties of RNA enzymes. Naturally occurring ribozymes can be divided into several groups based on their size: small self-cleaving RNAs ( \ 200 nucleotides), medium-sized self-splicing introns, and larger Review Focus on Function: Single Molecule RNA Enzymology Correspondence to: Nils G. Walter; e-mail: nwalter@umich.edu or David Rueda; e-mail: rueda@chem.wayne.edu ABSTRACT: The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays. # 2007 Wiley Periodicals, Inc. Biopolymers 87: 302–316, 2007. Keywords: single molecule microscopy; fluorescence resonance energy transfer; ribozyme; ribosome; catalytic RNA Contract grant sponsor: National Institutes of Health Contract grant number: GM62357 Contract grant sponsor: Wayne State University V V C 2007 Wiley Periodicals, Inc. 302 Biopolymers Volume 87 / Number 5–6