Raman and Raman optical activity (ROA) analysis of RNA structural motifs

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Vibrational Spectroscopy 48 (2008) 37–43 www.elsevier.com/locate/vibspec

Raman and Raman optical activity (ROA) analysis of RNA structural motifs Alison J. Hobro, Mansour Rouhi, Graeme L. Conn, Ewan W. Blanch * Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Greater Manchester M1 7DN, UK Received 26 October 2007; received in revised form 16 November 2007; accepted 21 November 2007 Available online 3 December 2007

Abstract Raman and Raman optical activity (ROA) spectroscopies are well suited to the study of ribonucleic acids (RNA) as they are both sensitive to a large amount of structural information and applicable to most RNAs and experimental conditions. As such, they can fill the current gap between high- and low-resolution techniques currently used by RNA biologists. In this study we illustrate the complementarity of Raman and ROA spectroscopies by their application to two functional RNAs, a 37 nucleotide RNA fragment from Domain I of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) RNA and the L11 binding domain ribosomal RNA (LBD rRNA). Fingerprint spectra unique to the structure of each RNA are presented. We then discuss the Raman and ROA spectra of several oligonucleotides containing three common secondary structural motifs all found within the EMCV RNA; the GNRA tetraloop, pyrimidine-rich asymmetric bulge and a base mismatch. We show that highly detailed structural information can be obtained from these spectra. # 2007 Elsevier B.V. All rights reserved. Keywords: Raman; Raman optical activity; EMCV IRES Domain I; L11 Binding domain rRNA; RNA structure

1. Introduction Our understanding of the functions of biological molecules is greatly advanced by our understanding of their structures. X-ray crystallography and high-resolution nuclear magnetic resonance (NMR) spectroscopy are the dominant techniques for structural elucidation but are often not applicable to biological molecules. Crystallography requires the growth of diffractable crystals, which is often difficult or impossible, while many molecules are too large for NMR studies. Vibrational spectroscopic techniques are playing an increasingly important role in structural biology as they are not only sensitive to a great deal of structural information but are also applicable to most biological molecules and under most conditions. Two particularly powerful vibrational techniques are Raman spectroscopy and Raman optical activity (ROA) [1–3]. ROA measures the small difference in the intensity of Raman scattering from chiral molecules in right- and leftcircularly polarized light or, equivalently, the small difference in the circularly polarized components of the Raman scattered light

* Corresponding author. Tel.: +44 161 306 5819; fax: +44 161 306 8918. E-mail address: [email protected] (E.W. Blanch). 0924-2031/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2007.11.007

[4,5]. Therefore, ROA measures the chirality associated with Raman transitions. Raman and ROA spectroscopies are now incisive structural probes of the structures of proteins and of macromolecular complexes such as viruses [2,3,6,7]. As Raman and ROA spectra can be measured simultaneously from the same sample, and are sensitive to different aspects of molecular structure, these data are complementary. This complementarity of Raman and ROA spectra has been exploited in studies by our group, and others, on proteins, with protein Raman spectra containing prominent bands from side chains that are sensitive to local environment and interactions, while protein ROA spectra are dominated by bands corresponding to secondary structural motifs and the tertiary fold. Raman and ROA have also been applied to nucleic acids but their combined potential for investigating ribonucleic acid (RNA) structure has not been fully explored. Raman spectroscopy was first applied to nucleic acids in the 1960s and has subsequently been used to investigate a wide range of natural and synthetic nucleic acids as well as their complexes with proteins, metal ions and drugs [3,8]. More recently, Raman spectroscopy has been used to probe some aspects of RNA structure, for example, to identify structural features and conformations such as the GNRA tetraloop [9–11]. Early

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studies on pyrimidine nucleosides [12] and polyribonucleotides [13–15] provided a starting point for the interpretation of ROA spectra of RNA. ROA can provide novel structural information about RNA as exemplified by a study of RNA molecules bound in cowpea mosaic virus (CPMV) capsids [7]. Although Raman and ROA are complementary techniques, they have generally only been used independently to study RNA structure. Here we demonstrate their combined application to probe RNA structure within two different RNA molecules: a 37 nucleotide RNA fragment from Domain I of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) RNA [16,17] and the L11 binding domain ribosomal RNA (LBD rRNA). The IRES RNA guides internal, non-cap-dependent, initiation of EMCV protein translation [18,19] and bases critical for IRES function, including a GNRA tetraloop structure, lie within this small fragment [20,21]. Significantly, no evidence for protein interaction with this domain has been observed implying that it is the RNA structure of Domain I itself that is vital for function [22]. This makes the domain an interesting target for the application of these two spectroscopic techniques as structural probes of a functional RNA and a good model system for establishing rigorous spectral analysis methods. The 37 nucleotide RNA fragment adopts a stable independently folded structure with the secondary structure motifs as shown in Fig. 1, and has been partially characterized structurally by NMR [23]. This provides both a basis for developing spectral interpretation and the opportunity to obtain new information about the environment of RNA bases in non-helical elements of the IRES Domain I secondary structure, and the contribution of these regions, individually and in combination, to the structure of the 37 nucleotide RNA fragment. The L11 domain of the bacterial ribosome is positioned at the base of a functionally important region of the 50S subunit called the L7/L12 stalk. There is increasing evidence, particularly medium resolution X-ray diffraction and lower

resolution cryo-electron microscopy structures [24–27], that the L11 domain is necessary for the binding and function of ribosome factors for initiation (IFs), elongation (EF-G) and release (RFs). It has been shown that the minimal binding site for the ribosomal protein L11 is a 58 nucleotide domain, called the LBD rRNA, from the 23S rRNA, shown in Fig. 1. The LBD rRNA is independently folded in isolation and without binding of the L11 ribosomal protein, and adopts complex secondary and tertiary structures. In this study we have investigated a stabilised version of the LBD RNA with mutation A1061. 2. Experimental 2.1. Sample preparation Three oligonucleotides were designed, corresponding to different secondary structure motifs of the 37 nucleotide RNA (shown in Fig. 1) and referred to as Hairpin RNA, Mismatch RNA and Bulge RNA, respectively: 1. A fully base paired helix with a GNRA (GCGA) tetraloop (Hairpin RNA). 2. Hairpin RNA incorporating a UC mismatch (A531 ! C change) (Mismatch RNA). 3. Hairpin RNA incorporating a pyrimidine-rich (AACCCCC) asymmetric bulge (Bulge RNA). Each oligonucleotide (chemically synthesised and HPLCpurified) was purchased from CureVac/VH Bio as lyophyilized powders. Further purification by preparative denaturing PAGE was found to improve the quality and consistency of spectra and was therefore used for all samples. The complete EMCV fragment and LBD RNAs were synthesised by in vitro transcription and purified by denaturing PAGE as described previously [23,28,29]. The EMCV RNA contains an additional 50 -G nucleotide (not shown in Fig. 1) to provide a strong

Fig. 1. The secondary structure of the 37 nucleotide domain from the EMCV IRES (left) and tertiary structure of the 58 nucleotide LBD RNA (right). The sequence of the EMCV IRES is shown along with those of three oligonucleotides containing structural features from the EMCV IRES. The Mismatch and Bulge RNAs are created by the addition of the single structural motif of interest (each feature is shown in a box), the UC mismatched base pair and pyrimidine-rich asymmetric bulge, respectively, to the Hairpin RNA. The EMCV RNA contains both of these motifs and all four RNAs contain the GNRA tetraloop. The tertiary structure of the LBD rRNA from the X-ray crystal structure of the RNA-protein complex (PDB file 1hc8 [40]) is shown to the right. The LDB RNA structure is the same in the absence of the L11 protein (M.S. Dunstan and G.L. Conn, unpublished data).

A.J. Hobro et al. / Vibrational Spectroscopy 48 (2008) 37–43

promoter for transcription and is therefore 38 nts in length. All samples were dissolved directly into 110 ml of buffer (50 mM MOPS, pH 6.5 with 150 mM KCl) to give final RNA concentrations in the range 19–35 mg/ml (1.6–2.8 mM), as described previously [30]. Each sample was centrifuged at maximum speed in a microcentrifuge before loading into a quartz microfluorescence cell and centrifuged again at low speed for 5 min in the cell to remove dust particles prior to spectroscopic analysis. 2.2. Spectroscopy All Raman and ROA spectra presented here were measured using a ChiralRaman Spectrometer (BioTools Inc.) [31] operating at 532 nm with laser power at the sample of 0.65 W, spectral resolution of 7 cm1 and total data accumulation times of 6–24 h. Raman and ROA spectra were acquired simultaneously for each sample. Although it was possible to measure high quality Raman spectra for these samples in less than 1 s, each Raman spectrum reported here was accumulated for the same time period as the corresponding ROA spectrum to ensure consistency of all data. The integrity of each RNA sample was assessed before and after data collection by denaturing acrylamide gel electrophoresis and ethidium bromide staining. No deterioration of any of the RNA samples was observed. 3. Results and discussion The Raman (top) and ROA (bottom) spectra for the EMCV and LBD RNAs are shown in Fig. 2. All Raman and ROA spectra presented in this paper underwent background

Fig. 2. Raman and Raman optical activity (ROA) spectra for the EMCV and LBD RNAs. Raman (top panel) and ROA (bottom panel) spectra are shown for the EMCV RNA (black) and LBD RNA (red). Raman spectra are presented as intensity sums (IR + IL) and ROA spectra as intensity differences (IR  IL). Spectra were normalized with respect to the intensity of the Raman band at 1098 cm1 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

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subtraction and intensity normalisation, to either experimental conditions or the Raman band at 1098 cm1, which is invariant of base composition, as previously described [30]. As the ROA spectra were collected simultaneously with their corresponding Raman spectra, the same normalisation ratios were applied to the ROA spectra as to the corresponding Raman spectra. There are a number of weak bands observed in all of these spectra between 300 and 700 cm1 but RNA Raman and ROA spectra are usually considered in terms of three structural origins. Raman and ROA bands in the region 700– 1150 cm1 originate in vibrations of the sugar rings and phosphate backbone. Bands in the region 1200–1550 cm1 principally arise from normal modes in which the vibrational coordinates of the base and sugar rings are mixed, while the region from 1550 to 1750 cm1 is dominated by bands characteristic of the bases and their stacking arrangements. Detailed analysis of the Raman and ROA spectra shown here for the EMCV and LDB RNAs will be furthered by comparisons with a greater library of spectra for functional RNAs than currently exists. The more extensive libraries of protein Raman and ROA spectra that already exist have allowed the application of chemometric methods to obtain new structural information [32,33], and in the future similar in silico analyses of Raman and ROA spectra may be used to investigate RNA structure. However, it is still possible at this stage to identify and compare characteristic band patterns for these two RNAs. In Fig. 2 (top panel), most of the Raman bands for the LDB RNA are more intense than those for the EMCV RNA, reflecting the greater size of the former (58 nucleotides compared to 38). Many of the bands for the two RNAs display similar shapes and identical positions, although relative band intensities vary due to differences in structure and sequence between the EMCV and LDB RNAs. Greater differences are observed between the two Raman spectra in the region from 1200 to 1400 cm1, and as described these bands probably originate in vibrational modes sampling both the conformations of ribose moieties in the ribose-phosphate backbone and also the interactions of the constituent bases. Therefore, these bands reflect differences in secondary and tertiary structures between these two RNAs. There are more obvious differences between the corresponding ROA spectra shown in Fig. 2 (bottom panel). Although the current library of ROA spectral features for RNA is limited, one relevant assignment is that for the negative– positive–negative triplet at 995, 1047 and 1083 cm1 associated with the C30 -endo ribose sugar pucker present in A-form double helices [7,13]. Both ROA spectra present this feature but the positive component of this triplet is obviously far more intense for the LDB RNA. Although the explanation for this difference is not yet fully explained, we believe that it reflects a more rigid and compact fold adopted by the LDB RNA. The observed differences in the positive ROA features at 814, 1321 and 1488 cm1 may also be associated with differences between tertiary structure. Comparison of the Raman and ROA spectra for the EMCV and LDB RNAs illustrates that these techniques can quickly differentiate between RNA structures and so provide unique

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spectral fingerprints. However, Raman and ROA can also provide far more detailed and quantitative information on RNA structure. Fig. 3 presents Raman (A, B and C top panels) and ROA (A, B and C bottom panels) spectra of the Hairpin, Mismatch and Bulge RNAs each compared alongside those of the EMCV RNA, which contains all three of these secondary

structure motifs. A summary of the observed Raman and ROA bands is shown in Table 1, with percentage differences for the intensity of each band for the Mismatch, Bulge and EMCV RNAs compared to those measured for the Hairpin RNA. We estimate that the relative Raman and ROA band intensities presented here are accurate to 5% for strong and medium bands. A detailed analysis of these spectra has been presented elsewhere [30]. A summary of these results is presented here, in which we describe the main effects of the UC mismatch and asymmetric pyrimidine-rich bulge secondary structure motifs on Raman and ROA bands. 3.1. Bands arising from ribose/phosphate/backbone

Fig. 3. Raman and Raman optical activity (ROA) spectra for Hairpin, Mismatch, Bulge and EMCV RNAs. Pairwise comparisons of Raman (top panel) and ROA (bottom panel) spectra for Hairpin RNA (black) and (A) Mismatch RNA, (B) Bulge RNA, and (C) EMCV RNA (red). The letters identifying each peak in the spectra correspond to those given in Table 1. Figure taken from Ref. [30]. Spectra were normalized with respect to the intensity of the Raman band at 1098 cm1 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.).

The Hairpin and Mismatch oligonucleotides are the same length and so have the same ribose and phosphate content, and consequently have similar Raman and ROA bands in this region. The same bands do show an increase in intensity for the Bulge and EMCV RNA spectra, relative to the Hairpin RNA spectrum, due to the addition of seven and eight residues, respectively, in these oligonucleotides. The Raman band observed at 813 cm1 originates from O–P–O stretching and is an indicator of A-form helical structure [3,9–11]. The introduction of a mismatched base pair is expected to disrupt the native A-type helix, leading to the decrease in intensity of this Raman band for the Mismatch RNA compared to that of the Hairpin RNA, and for the EMCV RNA compared to the Bulge RNA. A similar decrease in intensity is observed for the Raman band recorded at 1047 cm1, originating from P–O stretching [34], although the decrease in intensity for the EMCV RNA relative to the Bulge RNA is greater than for that observed for the Raman band at 813 cm1. The Raman band recorded at 1098 cm1, assigned to PO2 stretching [34] and as stated above used as an internal standard for intensity normalisation, shows a greater intensity in the spectrum of the Bulge RNA compared to that of the Hairpin and Mismatch RNA, with this increase due to the addition of the seven extra residues in the pyrimidine-rich bulge. Fewer band assignments currently exist for ROA than for Raman spectra of RNA, but most ROA bands observed from 950, or possibly lower, to 1150 cm1 are associated with the sugar-phosphate backbone. The intensities of the negative– positive–negative triplet at 994, 1049 and 1086 cm1 originating in the C30 -endo ribose sugar pucker in A-form helix suggest that the introduction of the UC mismatch one or more of the ribose sugars to be more constrained in terms of conformational mobility. It is important to note that ROA spectra are sensitive to conformational mobility and more rigid chiral structures generate more intense ROA spectral features [4]. Introduction of the pyrimidine bulge decreases the intensity of this triplet, monitoring a change in sugar pucker conformations, and probably a decrease in A-form helical content for the Bulge RNA. The largest intensity for this feature is observed for the EMCV RNA indicating that the presence of both the mismatch and bulge motifs in a sequence has a co-operative effect, resulting in a structure with more constrained C30 -endo sugar-pucker conformation than found for sequences only

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Table 1 Raman and ROA bands for the Hairpin, Mismatch, Bulge and EMCV RNAs Raman Band (cm1)a

Intensity relative to Hairpin (%) b

Assignment

Mismatch

Bulge

EMCV

Bands arising from ribose/phosphate/backbone c 601 wk Ribose [37] h 813 str O–P–O symmetric stretching, marker for A-form helix [3,9–11,38] i 856 wk Ribose-phosphate [37]. k 919 wk Ribose-phosphate, –C–O– stretching [37,39] l 977 wk Ribose-phosphate, –C–O– stretching [37,39] o 1047 str P–O stretch, sugar phosphate –C–O– stretching [34,39] p 1098 med PO2 symmetric stretching [3,9–11,38] q 1136 wk Stretching at 20 position of ribose [37]

+6 9

+30 +36

+55 +18

19 15 0 18 2 +5

68 5 +21 +38 +18 +71

56 0 +24 10 +21 +22

Bands arising from bases a 489 wk b 580 wk d 631 wk e 670 wk f 726 wk g 787 str j 876 wk

15 +16 +5 +4 21 +3 16

+35 +34 +49 13 +23 +47 50

+13 +13 +46 1 +4 +45 56

+9 +9 10 7 22 11 14 17 3 +78 6

24 +62 20 +12 +22 39 +66 +8 20 100 10

+32 +44 +12 +13 +12 22 +17 19 +3 +110 +1

+3 16

+16 +63

+26 0

+26 +14

16 19

+46 +2

r s t u v w x y z aa ab

1182 1253 1300 1316 1336 1380 1425 1460 1485 1532 1578

wk med sh med med/sh wk wk med str wk str

G out of plane ring deformations [39] A/U/G/C out of plane ring deformations [39] A/U/G/C [10,36,39] G out of plane ring deformations [9–11,37,39] A ring stretching [9,11,35,37,39] C/U breathing/stretching [35–37,39] A/U/G/C out of plane ring deformations, backbone [10,36,39] A/U/G/C ring; external C-N stretching [9,10,36,39] U/C ring stretching [3,9,11,35,37,39] C/A ring stretching [10,11,37,39] A/G ring stretching [3,9–11,37,39] A/G/U [3,9–11,37,39] A/G/U [10,11,37,39] A/G ring stretching, CH deformations [36,37,39] U/C ring stretching, CH deformations [3,9–11,37,39] A/G ring stretching/planar vibrations [9,10,36,37,39] A/G/C ring stretching [10,35–37,39] A/G ring stretching [9,10,35–37,39]

Bands with no previous assignment m 1002 wk n 1025 med/sh

ROA band (cm1) ac 994–1093 ad 1650–1750

Triplet; A-form RNA [13] Couplet; right handed helix [13]

Table adapted from Ref. [30]. a Letters identifying each peak which correspond to those given in Fig. 3. Band positions given are those observed in the spectrum of the Hairpin RNA, and may differ slightly for the other three oligonucleotides shown in Fig. 3. Intensity key: wk: weak, med: medium, str: strong, sh: shoulder. b The intensities for each of the other three oligonucleotides are given as percentages of the Hairpin RNA intensity for each band.

containing one of these two motifs. Cooperative effects on local RNA structure such as these are possible as the mismatch pair in EMCV RNA is located only three residues away from the pyrimidine-rich bulge (Fig. 1). 3.2. Bands arising from bases Intensities of the Raman bands observed at 489 cm1 and 726 cm1 are sensitive to the number of adenines in each oligonucleotide. The Raman band observed at 787 cm1 originates in breathing or stretching vibrational modes of cytosine and uracil bases [35,36] and differences in intensity of this band between the four oligonucleotides indicate that this band may provide direct information on the environment of these bases. Inclusion of a single cytidine nucleotide into the

A-form duplex, so forming the UC mismatched base pair, leads to a small increase of 3% in intensity, within experimental error. However, when the five cytidine bases of the pyrimidine-rich bulge loop are present, we observe an increase of 10% per residue in this band. This increase in intensity is significantly larger than the proportional increase in the cytosine/uracil content for both the Bulge RNA and EMCV RNA, showing that the Raman band at 787 cm1 is sensitive to the environment of the pyrimidine bases and particularly those in non-helical regions. Similarly, Raman bands at 1300, 1316 and 1336 cm1 reflect the number of adenine bases in each RNA, while the band at 1253 cm1 is associated with cytosine vibrations. The Raman band observed at 1380 cm1 also appears to directly reflect changes in the adenine content of the RNAs, but

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is also sensitive to the context in which the adenines are added to or removed from the sequence. The differences in intensity of this band between the Hairpin RNA and the Mismatch (11%) and Bulge (39%) RNAs are not fully reflected in the EMCV RNA (22%) suggesting that there are cooperative changes in the RNA structure upon combining multiple structural motifs, as may be expected. This type of behaviour is also observed for the Raman bands at 580, 1047, 1253 and 1300 cm1. The phenomena of Raman hyperchromism, where Raman band intensity increases with increased base stacking interactions [36], and Raman hypochromism, where there is a decrease in Raman band intensity with increased face-to-face stacking of the bases [37], can be a useful probe of RNA structure. Such changes in band intensity can be affected or induced by temperature, due to its influence on base stacking interactions [36,38]. The Raman bands measured at 1380, 1485 and 1578 cm1 in these spectra are hypochromic [9,35] and indicate that introduction of the pyrimidine bulge reduces the face-to-face base stacking of the RNA, and the observed decrease in band intensity. In the ROA spectra, the broad negative–positive couplet between 1600 and 1650 cm1 indicative of A-type helix [13] is present in all cases, though there is a reduction in intensity of this couplet for the Bulge RNA. This indicates that the introduction of the pyrimidine bulge significantly disrupts the A-form helical structure of the RNA as the presence of a large asymmetrical bulge loop is likely to produce a sharp turn in the RNA helix, separating and potentially unstacking the two helical regions on either side. The change in intensity of this ROA couplet between the cases of the Hairpin and Bulge RNAs appears to reflect the expected changes in RNA structure. In contrast, in comparing the ROA spectra for the Hairpin and Mismatch RNAs, where a UC mismatch has been incorporated, an apparent increase in A-form helical content is observed. This finding is also observed for the EMCV RNA, which also contains the pyrimidine-rich bulge but maintains an apparent helical content, where we observe a significantly greater intensity for this couplet than for the Hairpin RNA. It appears that, as with Raman spectra, ROA can monitor the presence of both motifs in a sequence and interaction between these two motifs. 4. Conclusion We have shown that Raman and ROA spectroscopies are able to reveal complementary, and detailed, information about RNA structure. In the first level of analysis, individual bands and combinations of bands provide a unique spectral fingerprint for each RNA that is highly sensitive to the tertiary structure. More detailed analysis of these spectra generates a wealth of information on the tertiary fold, secondary structure motifs such as the GNRA tetraloop, pyrimidine-rich bulge and single nucleotide mismatch studied here; and even the environments of specific bases. These Raman and ROA bands can serve as marker bands for secondary structure elements and also monitor interactions between these elements. Although Raman and ROA spectra

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