Raman crystallography of RNA
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.
References (73)
- et al.
Trends Biochem. Sci.
(2004) Curr. Opin. Struct. Biol.
(2005)- et al.
Biophys. J.
(2006) - et al.
Biochimie
(2006) - et al.
Curr. Opin. Struct. Biol.
(1999) - et al.
J. Mol. Biol.
(2000) - et al.
J. Mol. Biol.
(2002) - et al.
J. Mol. Biol.
(2004) - et al.
Methods Enzymol.
(2000) - et al.
Chem. Biol.
(2007)
Biophys. J.
Biophys. J.
Structure
Methods Enzymol.
Nature
Science
Nat. Rev. Mol. Cell. Biol.
Nat. Struct. Mol. Biol.
Science
Genes Dev.
Methods Mol. Biol.
Q. Rev. Biophys.
RNA
Science
Front. Biosci.
Methods Mol. Biol.
J. Am. Chem. Soc.
J. Am. Chem. Soc.
Biochemistry
Annu. Rev. Phys. Chem.
Annu. Rev. Phys. Chem.
Principles of Instrumental Analysis
Annu. Rev. Biophys. Biomol. Struct.
Cited by (29)
Surface-enhanced Raman spectroscopy for analysis of PCR products of viral RNA of hepatitis C patients
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :The peak at 1205 cm−1 can be correlated with the C-N stretching vibration of cytosine in the RNA [31]. On the other hand, the unique SERS spectral features seen in the mean SERS spectra of RNA of both healthy/control and HCV positive samples shown by solid lines, include those at 680 cm-1 (out of plane 5-member ring deformation adenine) [48], 729 cm-1 (peak of adenine), 1033 cm−1 (C-N stretching of ribose) and 1205 cm−1 (C-N stretching of cytosine), 1463 cm−1 (ribose vibration) [34], 1495 cm−1 (C-C bond stretching of cytosine) [41], 1528 cm−1 (guanine) [42], 1581 cm−1 (guanine, adenine peak) [49], 1630 cm- 1 (riboadenosine) [43]. Moreover, the peak intensities of certain spectral signatures at 729 cm−1, 952 cm−1, 1153 cm−1, 1205 cm−1, 1342 cm−1, and 1463 cm−1 are steadily increased due to increase in viral load values, whereas other characteristic features are found to have decreased in the intensity with the increasing viral load.
Multiscale methods for computational RNA enzymology
2015, Methods in EnzymologyCitation Excerpt :Divalent metal ions (usually Mg2+) are universally important for folding under physiological conditions (Grilley, Soto, & Draper, 2006; Misra & Draper, 2002) and, in ribozymes, have been implicated in many instances as directly participating in catalysis (J. Chen et al., 2013; Wong & York, 2012). This makes their roles in folding and catalysis difficult to untangle (Gong, Chen, Bevilacqua, Golden, & Carey, 2009; Gong, Chen, Yajima, et al., 2009; Misra & Draper, 1998). Similarly, the structure and energetics of RNA, in particular tertiary interactions, are sensitive to pH (Murray, Dunham, & Scott, 2002; Nixon & Giedroc, 2000; Wadkins, Shih, Perrotta, & Been, 2001), as are the catalytic protonation state requirements of key active site residues in ribozymes (Butcher & Pyle, 2011; Doudna & Cech, 2002).
Experimental approaches for measuring pK<inf>a</inf>'s in RNA and DNA
2014, Methods in EnzymologyCitation Excerpt :Here, we provide a brief summary of the Raman crystallography pKa method developed by the Carey, Golden, and Bevilacqua labs. We have provided a full methods paper on pKa determination of RNAs by Raman earlier and refer the interested reader to this (Gong et al., 2009). Crystals of the precleaved ribozyme were grown in the presence of a modification of the nucleophilic 2′OH to methoxy, deoxy, or fluoro to prevent ribozyme reactivity in the crystal.
Base ionization and ligand binding: How small ribozymes and riboswitches gain a foothold in a protein world
2011, Current Opinion in Structural BiologyKinetic crystallography by Raman microscopy
2011, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :RNA-based enzymes, ribozymes, provide very interesting comparisons and contrasts to protein enzymes making them a natural extension for Raman crystallographic analysis. Our work on ribozyme crystals has been reviewed recently and will not be repeated here [38]. However, Table 1, from Gong et al. summarizes the chemical information obtained using the HDV ribozyme as a paradigm.