%0 Journal Article
%T Probing Retroviral and Retrotransposon Genome Structures: The ¡°SHAPE¡± of Things to Come
%A Joanna Sztuba-Solinska
%A Stuart F. J. Le Grice
%J Molecular Biology International
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/530754
%X Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100¨C500£¿nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2¡ä hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide ¡°connectivity map,¡± conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges. 1. Introduction Cis-acting sequences within the (+) strand RNA genomes of retroviruses and long terminal repeat (LTR) containing retrotransposons control several critical events in their life cycle, including transcription [1], translation [2], dimerization [3], packaging [4], RNA export [5], and DNA synthesis [6]. Development of novel RNA-based strategies to ameliorate human immunodeficiency virus (HIV) pathogenesis would therefore benefit from an improved understanding of RNA structure and how this mediates interactions with both host and viral proteins. Historically, deciphering higher-order RNA structure has taken advantage of base- and structure-specific nucleases (e.g., RNases A, T1, T2 [7] and nuclease S1 [8]) or chemicals (e.g., dimethyl sulfate, diethyl pyrocarbonate [9, 10], and Pb2+ [11]). While these approaches have produced seminal advances in elucidating features of the HIV-1 and HIV-2 genomes [12¨C23], the necessity in most cases for multiple reaction conditions can be considered a limitation. Moreover, in almost all instances, enzymatic and chemical RNA footprinting has been performed on short RNAs prepared by in vitro transcription and labeled with 32P, eliminating any
%U http://www.hindawi.com/journals/mbi/2012/530754/