ACTA MEDICA LITUANICA. 2007. Vol. 14. No. 3. P. 165–169 © Lietuvos mokslų akademija, 2007 © Lietuvos mokslų akademijos leidykla, 2007 © Vilniaus universitetas, 2007 RNA interference: from a research tool to a novel therapeutic agent RNA interference (RNAi) is a powerful and highly specific gene-silencing approach that is trig- gered by the formation of double-stranded RNA helices and directs silencing of gene expression in a sequence specific manner. RNAi is an evolutionarily conserved natural mechanism that plays an important role in maintaining the genomic integrity of eukaryotic organisms, con- trolling gene expression, and guarding against exogenous viral infection. Over the past decade, this ancient cellular response has been exploited as an invaluable research tool for functional characterization of known genes, aided in identification of genes with unique functions, and has evolved as a novel approach for post-transcriptional gene silencing with potential clinical appli- cations. In this review we will briefly highlight our current understanding of the RNAi process and discus how the use of RNAi technology has facilitated the advent of major discoveries in functional genomics and development of novel gene therapies. Key words: RNA interference, gene-silencing, gene therapy Jolanta Vidugirienė 1 , Said Goueli 1 , Kęstutis Sužiedėlis 2 1 Department of Cell Analysis, Promega Corporation, USA 2 Institute of Oncology, Vilnius University, Lithuania INTRODUCTION Over the past decade, RNA interference has emerged as a natural mechanism for silencing gene expression (1, 2). Te phenome- non was first noticed in plants, when attempts to enhance the violet color of petunias by introducting extra copies of pigment gene resulted in an unexpected decrease in gene expression and the appearance of several white flowers (3). However, these and similar observations remained an enig- ma until it was reported by Fire and Mello (2006 recipients of the Nobel Prize in Physiology or Medicine) (4) that double-stranded RNA molecules injected into nematode worm C. elegans di- rected the degradation of messenger RNA in a sequence spe- cific matter. Tis sequence-specific degradation of messenger RNA (mRNA) was coined RNA interference (RNAi) and since injection of small amounts of double-stranded RNA was effica- cious, it has been proposed that RNA interference is a catalytic process. Te discovery of new enzymatic machinery in cells raised several fundamental questions to be addressed. What is the natural role of RNAi process? What is the cellular structure and function of the gene silencing machinery? And more important- ly, does this mechanism exist in other organisms and can it be used for specific gene silencing? During the next few years the molecular mechanism in- volved in handling dsRNA molecules has been extensively re- searched. In a very short time RNAi became a “powerful new Correspondence to: J. Vidugirienė, Department of Cell Analysis, Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA. E-mail: jvidugiriene@promega.com tool” for functional genomic analysis, target validation and gene knockdown in cell and animal models. Te main com- ponents of RNAi enzymatic machinery have been uncovered, and the important natural role of RNAi process has been elu- cidated. RNAI SILENCING MECHANISM Depending on the organism, RNAi can be triggered by various sources of dsRNA molecules, including long dsRNAs, exogenous- ly introduced small dsRNAs known as siRNAs or plasmid-based short hairpin RNAs (shRNAs) (Fig. 1). In cells, long dsRNAs and hairpin RNAs are recognized by an endonuclease Dicer and are converted into short-interfering RNAs (siRNAs) with 2-nt over- hangs at the 3' ends and phosphate groups at the 5' ends (5, 6). Small interfering siRNAs generated by Dicer or introduced ex- ogenously are loaded into RNA-induced silencing complexes (RISCs). RISC complex unwinds the double-stranded siRNA and preferentially utilizes one of the RNA strand as a guide to tar- get complementary RNA molecules for degradation (7–9). Te minimal defining features of RISC include the presence of an Argonaute family member and the guide strand of a small RNA. Te guide-strand-containing RISC complex then binds to the corresponding mRNA and directs it for cleavage which is carried out by the catalytic domain of Argonaute 2 (10–12). Te 5' end of the guide strand sets the location of the cleavage site for target- RNA and the cleavage which occurs between 10 and 11-nt up- stream of the 5' end. Te cleaved messenger RNA is targeted for degradation, while RISC complex is recycled and can undergo multiple rounds of mRNA cleavage (for more information see recent reviews 13–16).