Elsevier

Methods

Volume 49, Issue 2, October 2009, Pages 101-111
Methods

Raman crystallography of RNA

https://doi.org/10.1016/j.ymeth.2009.04.016Get rights and content

Abstract

Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

Introduction

RNA is involved in myriad biological function and controls gene expression at almost all levels. The leading models for the origin of life posit that life began with a so-called ‘RNA World’, in which RNA served genetic, structural, and functional roles [1], [2], [3]. Such biological complexity necessitates molecular complexity. Indeed, it was demonstrated more than 30 years ago that tRNA can adopt complex folds with extensive non-Watson–Crick base pairing, intricate helical stacking, and diverse metal ion binding interactions [4], [5].

In the ensuing years, functional RNAs have been discovered that catalyze chemical reactions (ribozymes) [6], [7] and bind small molecules and alter gene expression (riboswitches) [8], [9], [10]. Over the past 15 years in particular, atomic-level experimental structures of ribozymes, riboswitches, and the ribosome have been determined and reveal intricate structures with buried active sites [11]. Most of these structures have been solved by X-ray crystallography at −170 °C, with contributions from solution NMR spectroscopy [12]. In addition, NMR spectroscopy and molecular dynamics (MD) are revealing how dynamics contribute to RNA function [13], [14]. An important goal in the field of RNA biology is to relate three-dimensional structures and dynamics to biological function.

A variety of solution studies, both in bulk solution and at the single molecule level, have revealed that a given RNA sequence can adopt multiple, stable structures and only a fraction of these can be functional (e.g. can bind substrates and catalyze chemical reactions) while others are non-functional [15], [16]. For example, rate-pH profiles of the HDV ribozyme showed that certain constructs have multi-exponential kinetics, which reflect 2 or more populations of the ribozyme only one of which has direct catalytic potential [17], [18], [19], [20]. In addition, single molecule studies on the hairpin ribozyme revealed four distinct species, which have ‘memory effects’ and do not interconvert [21]. The tendency of RNA to misfold also applies to larger RNAs [22]. These properties indicate that structural and functional studies of RNA must be reconciled.

Ribozymes provide an ideal vehicle for assessing RNA folding because their native states have one or more specific, testable functional properties, including metal ion binding, proton binding, and bond cleavage. Because RNA tends to crystallize in a single conformation, crystallography offers the potential to isolate and describe single ribozyme states [23]. However, determining whether these states are functionally relevant has proven challenging, in part because crystals are typically studied under cryo-conditions near 100 K, which limits dynamics and the ability to perform biophysical experiments, and also because modifications are often needed to keep RNAs from reacting in the crystal, which can be perturbing. In an effort to characterize the functional properties of RNA crystals, we have applied Raman spectroscopy to single crystals of a small ribozyme under ambient conditions in the hanging drop [24], [25], [26]. This article will focus entirely on RNA and primarily on one crystal form of a small ribozyme from the authors’ laboratories and on a study on tRNA. Although these are the only studies available in the literature, the methodology is also applicable to proteins, with many examples from the Carey lab [27]. The generality of Raman crystallography of RNA will be determined as additional RNA systems are examined in the future.

Section snippets

Advantages and disadvantages of Raman crystallography for characterizing RNA

In this section we consider some of the advantages and disadvantages of Raman spectroscopy versus other spectroscopies for characterizing RNA, followed by some of the advantages and disadvantages of working with crystals.

Chemical information obtainable by Raman crystallography of RNA

One of the main advantages of Raman spectroscopy is that it allows unique and extensive chemical information to be obtained about RNA (summarized in Table 1). This is best illustrated through consideration of literature on Raman crystallography of RNA. In the first subsection, we consider the early, albeit limited, Raman studies of RNA crystals and the molecular insight into RNA that was gained. In the second subsection, we describe some of our recent Raman studies of RNA crystals and

Sample preparation

This section is concerned with the design, synthesis, purification, and crystallization of RNA samples for Raman crystallography. Each of these topics has been extensively reviewed in the literature and so is summarized only briefly here, with issues relevant to Raman crystallography emphasized.

The Raman microscope

In the 1980s and 1990s, the method of Raman microscopy was further developed wherein an optical microscope is coupled to a Raman spectrometer to collect Raman scattering from micron-sized samples [27]. The Raman crystallography setup used in our laboratory is depicted in Fig. 1 and summarized in the right-hand column of Table 2. A krypton ion laser (Coherent Innova 70C; Coherent, Inc.) produces a 647.1 nm red light (see first horizontal red line from top in Fig. 1), which scatters from the RNA.

Conducting Raman experiments

Obtaining adequate Raman data on RNA requires attention to a number of experimental details. In this section, we consider the major steps involved in conducting Raman experiments including instrument calibration; crystal preparation; soaking of ligands; acquisition, processing and interpretation of Raman data; and measurement of physicochemical properties of RNA. Typical experimental parameters both for the Raman microscope system and the RNA crystal are presented in Table 2. These parameters

Concluding remarks

Raman crystallography has provided unique insights into RNA physicochemical properties, including pKa’s near neutrality; metal ion binding mode, quantification, affinity, and ligands; and RNA conformational changes. Where comparisons are possible, agreement between biochemical and Raman crystallography has been superb. Native folding of the HDV ribozyme in solution and in the crystal, with its high solvent content, likely gives rise to this agreement. Based on this, we are encouraged that

Acknowledgments

P.C.B.: NSF Grant MCB-0527102 and the Pennsylvania State University for sabbatical support. B.L.G.: Purdue University Department of Biochemistry, and the Purdue University Cancer Center. P.R.C.: NIH Grant GM-54072.

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