Structures of CUG Repeats in RNA
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m202235200
ISSN1083-351X
AutoresPhilip Pinheiro, Garry P. Scarlett, Alison Rodger, P. Mark Rodger, Anna Murray, Tom Brown, Sarah F. Newbury, James A. McClellan,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoTriplet repeats that cause human genetic diseases have been shown to exhibit unusual compact structures in DNA, and in this paper we show that similar structures exist in shorter “normal length” CNG RNA. CUG and control RNAs were made chemically and byin vitro transcription. We find that “normal” short CUG RNAs migrate anomalously fast on non-denaturing gels, compared with control oligos of similar base composition. By contrast, longer tracts approaching clinically relevant lengths appear to form higher order structures. The CD spectrum of shorter tracts is similar to triplex and pseudoknot nucleic acid structures and different from classical hairpin spectra. A model is outlined that enables the base stacking features of poly(r(G-C))2·poly(r(U)) or poly(d(G-C))2·poly(d(T)) triplexes to be achieved, even by a single 15-mer. Triplet repeats that cause human genetic diseases have been shown to exhibit unusual compact structures in DNA, and in this paper we show that similar structures exist in shorter “normal length” CNG RNA. CUG and control RNAs were made chemically and byin vitro transcription. We find that “normal” short CUG RNAs migrate anomalously fast on non-denaturing gels, compared with control oligos of similar base composition. By contrast, longer tracts approaching clinically relevant lengths appear to form higher order structures. The CD spectrum of shorter tracts is similar to triplex and pseudoknot nucleic acid structures and different from classical hairpin spectra. A model is outlined that enables the base stacking features of poly(r(G-C))2·poly(r(U)) or poly(d(G-C))2·poly(d(T)) triplexes to be achieved, even by a single 15-mer. Trinucleotide repeat expansion diseases (TREDs) 1The abbreviations used are: TRED, trinucleotide repeat expansion disease; NOE, nuclear Overhauser effect. 1The abbreviations used are: TRED, trinucleotide repeat expansion disease; NOE, nuclear Overhauser effect. are genetic disorders that are caused by expanded tracts of repeated sequences (usually CNG) (reviewed in Ref. 1Warren S.T. Nelson D.L. Curr. Opin. Neurobiol. 1993; 3: 752-759Crossref PubMed Scopus (47) Google Scholar). Naturally variable lengths of these tracts occur in several genes involved in neurological disorders. Up to about 30 trinucleotide repeats do not cause neurological defects, but once a certain critical length is exceeded, disease ensues. The disease-causing alleles are usually dominant. The TREDs are complex syndromes that may exhibit anticipation (genetic instability leading to longer tract lengths with each generation). In several cases there is evidence that the longer the tract, the more severe or the earlier the onset of disease. Proof that long tracts cause disease rather than merely being correlated with it was obtained by artificially introducing long CNG tracts into mice, thereby inducing a version of spinocerebellar ataxia type I (2Burright E. Clark H. Servadio A. Matilla T. Feddersen R. Yunis W. Duvick L. Zoghbi H. Orr H. Cell. 1995; 82: 937-948Abstract Full Text PDF PubMed Scopus (517) Google Scholar). In those experiments some, but not all, features of the syndrome were reproduced in the mouse model, and in fact it is known that even in humans, instability and other features of the syndromes depend on poorly understood contributions of the genetic background (3Goldberg Y. McMurray C. Zeisler J. Almqvist E. Sillence D. Richards F. Gacy A. Buchanan J. Telenius H. Hayden-M R. Hum. Mol. Genet. 1995; 4: 1911-1918Crossref PubMed Scopus (111) Google Scholar). Although long tracts cause disease, several lines of evidence suggest that the shorter tracts perform some useful function; for example, CNG tracts in neurodevelopmental genes seem to have grown during primate evolution (4Djian P. Hancock P. Chana H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 417-421Crossref PubMed Scopus (58) Google Scholar). There are three main outstanding questions regarding the TREDs: (i) what is the normal function of the CNG tracts?, (ii) why and how does the long tract cause disease?, and (iii) is it correct to talk of the TREDs as a unitary phenomenon, or should we be distinguishing Fragile X and myotonic dystrophy (where the CGG or CTG repeats are normally transcribed but not translated) from Huntington's Chorea, SCA1, and other diseases where the CAG repeats are translated into polyglutamine, resulting in proteins that differ from wild-type protein (5Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1876) Google Scholar, 6Li X.-J., Li, S.-H. Sharp A.H. Nucifora F.C.J. Schilling G. Lanahan A. Worley P. Snyder S.H. Ross C.A. Nature. 1995; 378: 398-402Crossref PubMed Scopus (531) Google Scholar, 7Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Holenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 8Trottier Y. Lutz Y. Stevanin G. Imbert G. Devys D. Cancel G. Saudou F. Weber C. David G. Tora L. Agid Y. Brice A. Mandel J.-L. Nature. 1995; 378: 403-406Crossref PubMed Scopus (578) Google Scholar)? Answers to these questions require molecular analysis of the differences between the long and short tracts. Accordingly, work in various laboratories has investigated the effects of the triplet repeats on various macromolecules as discussed below. Even when the triplet repeats are translated, the protein by itself cannot explain even the CAG TREDs, because if it did there would be a class of CA(Purine) diseases (CAA is another codon for glutamine). Furthermore, in the case of Fragile X it appears that absence of the protein and/or RNA in the cytoplasm causes the main features of the disease (9Kooy R.F. Willemsen R. Oostra B.A. Mol. Med. Today. 2000; 6: 193-198Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). There are several mechanisms that can lead to this result. One possibility is that expanded-tract alleles of FMR-1 are hypermethylated (perhaps because CGG repeats are particularly good substrates for methyltransferases; (10Smith S.S. Laayoun A. Lingeman R.G. Baker D.J. Riley J. J. Mol. Biol. 1994; 243: 143-151Crossref PubMed Scopus (81) Google Scholar)), and this is associated with genetic shutdown. That absence of gene function can explain the condition is shown by the existence of rare deletions in FMR-1, leading to symptoms of Fragile X (11Gu Y. Lugenbeel K.A. Vockley J.G. Grody W.W. Nelson D.L. Hum. Mol. Genet. 1994; 3: 1705-1706Crossref PubMed Scopus (40) Google Scholar, 12Lugenbeel K.A. Peier A.M. Carson N.L. Chudley A.E. Nelson D.L. Nature Genet. 1995; 10: 483-485Crossref PubMed Scopus (114) Google Scholar, 13Trottier Y. Imbert G. Poustka A. Fryns J.-P. Mandel J.-L. Am. J. Med. Genet. 1994; 51: 454-457Crossref PubMed Scopus (35) Google Scholar). Interestingly, intermediate-sized alleles (41–60 repeats) of the tract in FMR1 are associated with learning difficulties in boys, despite the fact that other symptoms of Fragile X are absent and that protein levels are normal (14Murray A. Youings S. Dennis N. Latsky L. Linehan P. McKechnie N. MacPherson J. Pound M. Jacobs P. Hum. Mol. Genet. 1996; 5: 727-735Crossref PubMed Scopus (141) Google Scholar). However, the totality of the syndrome also includes a DNA effect: the expanded-tract chromosomes themselves are physically fragile and genetically hypervariable. This may be a general property of CNG tracts, since these are known to mediate genetic instability not only in eukaryotes but also in Escherichia coli(16Darlow J. Leach D. Genetics. 1995; 141: 825-832Crossref PubMed Google Scholar, 17Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (147) Google Scholar, 18Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 2G. Scarlett and P. Pinheiro, manuscript in preparation. 2G. Scarlett and P. Pinheiro, manuscript in preparation. Similar behavior of other repeated sequences was previously shown to reflect cellular reaction to the in vivo formation of non-B DNA secondary structures (19Greaves D. Patient R. Lilley D. J. Mol. Biol. 1985; 185: 461-478Crossref PubMed Scopus (130) Google Scholar, 20McClellan J.A. Boublikova P. Palecek F. Lilley D.M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8373-8377Crossref PubMed Scopus (150) Google Scholar). Less attention has been paid to the role of the RNA. Nevertheless, since in all cases the CNG tracts are transcribed but in the loci associated with the two commonest diseases (Fragile X and myotonic dystrophy), they are not translated, it is likely that effects at this level are of central importance, at least for the normal function of the tracts (21Hamshere M.G. Brook J.D. Trends Genet. 1996; 12: 332-334Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 22McClellan J.A. Newbury S.F. Nature. 1996; 379: 396Crossref PubMed Scopus (1) Google Scholar). As for how the expanded tracts actually cause disease, the complex nature of the syndromes suggests that this is likely to involve all these aspects. For example, in cases where the tracts are translated, the production of mutant protein may have effects on the cell; if the expanded tracts form a novel structure in DNA, this may act to stimulate recombination or to compromise replication, thus accounting for anticipation and chromosomal fragility; and formation of a new structure in RNA may lead to abnormal stability, splicing, localization, or translation. The clearest candidate for a CNG disease mediated largely by effects at the RNA level is myotonic dystrophy (21Hamshere M.G. Brook J.D. Trends Genet. 1996; 12: 332-334Abstract Full Text PDF PubMed Scopus (12) Google Scholar). The shortest repeats that are known from the general population are about 15 nucleotides in length (five repeats). The shorter abnormal alleles are associated with adult-onset myotonic dystrophy and the longer ones with the congenital form. In this case a CTG repeat (CUG in the RNA) is untranslated and located in a locus involved in neuromuscular development. The disease-causing allele is dominant. Long RNA containing the expanded tracts has been shown to be transcribed effectively and to accumulate in foci within the nucleus (23Taneja K.L. McCurrach M. Schalling M. Housman D. Singer R.H. J. Cell Biol. 1995; 128: 995-1002Crossref PubMed Scopus (482) Google Scholar). Our working hypothesis therefore is that there are special features of CNG RNA structure or biochemistry. These special features mediate some useful function in the short, wild-type alleles and possibly also a deleterious function in the long, disease-causing alleles. In this paper we have used in vitro transcription and chemical synthesis to produce CUG RNA of various sizes and compared it with control RNAs, such as GUC (different polarity) and a randomized but isobasic sequence, using gel electrophoresis, circular dichroism (CD) spectroscopy, and thermal melting of the RNA. Plasmids were propagated in Escherichia coli DH5α, and supercoiled DNA was prepared by alkaline lysis and purified by ultracentrifugation in caesium chloride (Invitrogen) gradients that contained ethidium bromide (Sigma). Long CNG tracts and RNA markers were produced by in vitro transcription of linearized plasmids pGEM1, pGEM2, and Bluescript KS+ (all originally from Promega). Plasmid Bluescript KS+ was linearized with restriction enzymesTsp501I, MwoI, HaeIII, andXbaI (all New England Biolabs) to produce markers that were 9, 24, 30, and 39 nucleotides long, respectively. Plasmids pGEM2 and pGEM1 were digested with MwoI and AluI, respectively. Transcription of these templates produced markers that were 16 and 18 nucleotides long. All transcriptions were performed on ∼1 μg of appropriately linearized plasmid in a 20-μl volume (5× T7 buffer (200 mm Tris-HCl, pH 7.9, 30 mmMgCl2, 10 mm spermidine) and 100 mm dithiothreitol (Promega), RNAguard (AmershamBiosciences), 2.5 mm cold nucleotides A, G, and U, and 100 μm cold CTP (Amersham Biosciences), T7 RNA polymerase (Promega), RNase free water (Sigma)) containing 1 μl (1 μCi; 0.33 nmol) of radioactive [α-32P]CTP (3000 Ci/mmol: ICN-FLOW). To remove the template the transcription reactions were treated with 2 μl of 0.1 mg/ml DNase I (RNase-free; AmershamBiosciences) and finally purified by phenol extraction and ethanol precipitation. DNA templates for transcription were constructed using oligonucleotides synthesized on an Applied Biosystems machine using phosphoramidate chemistry. For efficient transcription it turned out to be crucial that each RNA start with a G, and to aid annealing a G + C-rich clamp was required at the “upstream” end of the duplex promoter region. In each case a top strand comprising the T7 promoter sequence was annealed to a bottom strand of which the 3′ end was its complement, and the 5′ end provided a template for the transcription of the following RNAs: G(CUG)5, G(CUG)6, and G(CUG)7. Control templates were constructed similarly, and they encoded G(GUC)5 or an isobasic control RNA 5′-GUGGUUGGCCUCCGUC-3′. DNase I treatment, phenol extraction, and ethanol precipitation were performed after transcription to remove the template. The sequences used are as follows: top strand of T7 promoter, 5′-GCCGGTAATACGACTCACATA-3′; template for transcribing G(CUG)5, 5′-(CAG)5CTATAGTGAGTCGTATTACCGGC-3′; template for transcribing G(CUG)6, 5′-(CAG)6CTATAGTGAGTCGTATTACCGGC-3′; template for transcribing G(CUG)7: 5′-(CAG)7CTATAGTGAGTCGTATTACCGGC-3′; template for transcribing G(GUC)5, 5′-(GAC)5CTATAGTGAGTCGTATTACCGGC-3′; template for transcribing isobasic control RNA, 5′-GACGGAGGCCAACCACTATAGTGAGTCGTATTACCGGC-3′; synthetic RNA for control purposes, 5′-(r(CUG)6)-3′. RNA markers and triplet repeat RNA transcripts were run on 10% non-denaturing and denaturing (7 m urea (Sigma)) acrylamide (29:1 acrylamide to bisacrylamide (National Diagnostics)) gels. In all cases the electrophoresis buffer was 1× TBE (90 mm Tris, pH 8.0 (Sigma), 90 mm boric acid (Sigma), 2 mm EDTA (Sigma)). Gels were fixed in 10% acetic acid and dried under vacuum onto Whatman 3MM paper, before autoradiography using Kodak film. DNA oligonucleotide markers 8–32 were purchased from AmershamBiosciences and labeled using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (3000 Ci/mmol; PerkinElmer Life Sciences). Synthetic RNA was labeled in a similar manner. RNA and DNA oligonucleotides for both CD and UV melting were dissolved to a concentration of 4 μm (for DNA) or 8 μm(for RNA) in 5 mm sodium phosphate buffer, pH 7.5, unless otherwise stated. All solutions for both the CD and UV melting experiments were filter-purified using a 0.2-μm nylon filter (Sigma). CD spectra were gathered on a Jasco J-715 CD spectropolarimeter using a 5-mm path length cell. Data were stored using the supplied software and then exported to Kaleidagraph for manipulation. Data were collected over the range 350–200 nm with a 0.5 nm resolution and 1 nm bandwidth at a speed of 100 nm/min; spectra were averaged over 16 scans. For comparison purposes the RNA CD data have been normalized to 1.2 OD260, while the DNA CD spectra have been normalized to 0.6 OD260. UV melting was conducted on a Cary 1 spectrophotometer ramped at 1 degree/min and the data collected with the supplied software, before exporting to Kaleidagraph for manipulation. Prior to the melting curve determinations, the samples were heated to 95 °C and cooled slowly (24Puglisi J.D. Tinoco I.J. Methods in Enzymol. 1989; 180: 304-325Crossref PubMed Scopus (653) Google Scholar). Melting curve measurements were repeated at least three times, and no significant differences were found between each set of data. The data were analyzed following Marky and Breslauer (25Marky L.A. Breslauer K.J. Biopolymers. 1987; 26: 1601-1620Crossref PubMed Scopus (1097) Google Scholar). The fraction of unfolded RNA in solution was determined by calculating, α=xx+yEquation 1 where x is the difference between the final absorbance of the unfolded RNA and the absorbance at a given temperature, and y is the difference in absorbance between the temperature-dependent absorbance and the base line corresponding to the temperature dependence of the unmelted RNA absorbance. The midpoint of the plot of α versus1/T, where T is the absolute temperature (in Kelvin), gives the melting temperature (by definition the temperature at which half of the RNA is melted). The van't Hoff transition enthalpy of the unimolecular transition is, ΔHyH=B′(1/Tmax)−(1/T2)Equation 2 where B′ = 3.50 cal K−1mol−1 (25Marky L.A. Breslauer K.J. Biopolymers. 1987; 26: 1601-1620Crossref PubMed Scopus (1097) Google Scholar), T max is the absolute temperature of the maximum of the α versus 1/Tplot, and T 2 is the absolute temperature of the high temperature half-height of the curve. The entropy of the transition is given as follows. ΔS=ΔHVHTmEquation 3 A variety of techniques were used to perform the conformational search within CHARMm version 25.2. These methods included a grid search based on the backbone torsion angles, a Boltzmann jump algorithm, and molecular dynamics simulations at temperatures of both 300 and 600 K. NOE constraints were used in some of the calculations to improve the chance of locating suitable Watson-Crick C-G base pairings within each triplet and also to locate possible stacked triplets; other calculations were performed without constraints. In all cases, the selected conformations were energy minimized at the end of the conformational searches; where NOE constraints were imposed the minimization was initially done with constraints imposed, but then subsequently with the constraints removed. The mobility of end-labeled chemically synthesized (CUG)6 electrophoresed on denaturing gels together with RNA markers (Fig.1A) shows that the RNA migrates as expected for an 18-mer. Electrophoresis of CUG repeat RNAs and control RNAs on denaturing gels shows that in vitro transcription generates bands of the expected sizes for all the samples (Fig.1 C). When these CUG repeat RNAs are electrophoresed on non-denaturing gels (Fig. 1, B and D), they migrate significantly faster than the marker RNAs. Randomized and GUC repeat RNAs migrate similarly to controls. The overall conclusion to be drawn from Fig. 1, A–D, is that the CUG RNAs are all unusually compact, whether they are made by chemical synthesis orin vitro transcription and that over this size-range their degree of compaction increases with tract length. This is in accord with the behavior previously observed for single-stranded DNA (26Mitchell J.E. Newbury S.F. McClellan J.A. Nucleic Acids Res. 1995; 23: 1876-1881Crossref PubMed Scopus (44) Google Scholar). For all the CUG and GUC repeats, it is interesting to note that extra bands at low molecular weight are observed. These extra small bands are not observed for the randomized control and probably reflect abortive transcription. DNA polymerase apparently also appears to pause at CNG tracts (27Kang S. Ohshima K. Shimizu M. Amirhaeri S. Wells R.D. J. Biol. Chem. 1995; 270: 27014-27021Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). 3A. Murray, unpublished observations. In addition to bands that move faster than the appropriate markers, some that move more slowly, especially in the six-repeat (19 nucleotides) and seven-repeat (22 nucleotides) tracks, are observed. These are not present in the denaturing gel nor in the chemically synthesized sample, so they are probably higher order complexes of RNA; possibly DNA/RNA hybrids that resist DNase I treatment. Multistrand complexes of CNG DNA have been observed previously by other workers (28Smith G.K. Jie J. Fox G.E. Gao X.L. Nucleic Acids Res. 1995; 23: 4303-4311Crossref PubMed Scopus (66) Google Scholar). Structural work on triplet repeats has been on short tracts that cannot be directly compared with those from TRED patients. Recently we have managed to create RNAs of lengths approaching clinical relevance by in vitro transcription of a range of plasmids containing 24–51 CTG repeats by cloning CNG tracts into bacterial plasmids under conditions where phenomena similar to anticipation may be observed.2 The gel mobility of these RNAs is shown in Fig. 1, E and F. These in vitrotranscriptions are not marked by the high levels of premature termination seen for the oligonucleotide templates. On the non-denaturing gel (Fig. 1 F) the CNG RNAs migrate as two bands which migrate anomalously slowly (in contrast to the anomalously fast migration of the smaller triplet repeats). Comparison with the mobilities of the markers shows this to be consistent with duplex or higher formation. Some sort of structural transition appears to occur between the 5–7 range and the 36–50 repeat range. Since this structural transition occurs at a length close to the threshold for myotonic dystrophy, this is potentially a result of some significance. To investigate the structures of the unusual compact structures found in the gels, CD spectra were collected for the chemically synthesized (CTG)5 and (CUG)5 samples and randomized controls. The CD spectrum of (CTG)5 in phosphate buffer consists of a positive peak at 285 nm and two negative peaks at 258 and 208 nm (Fig. 2 A). The 285 nm maximum is at too long a wavelength for the molecule to be adopting an A-DNA conformation, and the spectra do not contain a positive peak at around 205 nm, which would be expected if the tract were adopting duplex B-DNA structure (29Park Y.W. Breslauer K.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6653-6657Crossref PubMed Scopus (100) Google Scholar, 30Gray D.M. Ratcliff R.L. Vaughan M.R. Methods Enzymol. 1992; 211: 389-405Crossref PubMed Scopus (248) Google Scholar, 31Scaria P.V. Shafer R.H. J. Biol. Chem. 1991; 266: 5417-5423Abstract Full Text PDF PubMed Google Scholar). The negative peak at 208 nm suggests that the tract has triplex character, as a negative peak between 200 and 220 nm is considered a hallmark of triplex DNA (29Park Y.W. Breslauer K.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6653-6657Crossref PubMed Scopus (100) Google Scholar, 30Gray D.M. Ratcliff R.L. Vaughan M.R. Methods Enzymol. 1992; 211: 389-405Crossref PubMed Scopus (248) Google Scholar, 32Radhakrishnan I. Patel D.J. Biochemistry. 1994; 33: 11405-11416Crossref PubMed Scopus (228) Google Scholar). Spectra for triplexes are usually able to be approximated as the sum of the CD for the component duplex and single-stranded DNA plus some extra intensity due to the more rigid structure (33Chastain M. Tinoco I.J. Nucleic Acids Res. 1992; 20: 315-318Crossref PubMed Scopus (21) Google Scholar). In accord with this the observed spectrum closely resembles that expected for the sum of poly(d(G-C))2 and poly(d(T)) (34Johnson K.H. Gray D.M. Morris P.M. Sutherland J.C. Biopolymers. 1990; 29: 325-333Crossref PubMed Scopus (12) Google Scholar). Addition of 20 mm NaCl or 20 mm MgCl2 increases the 285 nm and 258 nm bands to the same intensity and increases the 208 nm band. The 208 nm band is particularly enhanced by MgCl2, which probably correlates with a stabilization of base stacking and more effective reduction of phosphate-phosphate repulsion (35Schweisguth D.C. Chelladurai B.S. Nicholson A.W. Moore P.B. Nucleic Acids Res. 1994; 22: 604-612Crossref PubMed Scopus (29) Google Scholar, 36Laing L. Draper D.E. J. Mol. Biol. 1994; 237: 560-576Crossref PubMed Scopus (126) Google Scholar, 37Cole P.E. Yang S.K. Crothers D.M. Biochemistry. 1972; 11: 4358-4368Crossref PubMed Scopus (207) Google Scholar). To investigate whether the observed triplex-like structure was inter or intramolecular, labeled (CTG)5 was titrated with unlabeled (CTG)5. Gel electrophoresis showed that there is no evidence for intermolecular structures (Fig. 1 G), which would be expected to shift the band up the gel due to an increased mass of the complex. There was also no evidence of concentration dependence of spectra other than that required by the Beer-Lambert Law. We therefore conclude that (CTG)5 forms a monomolecular base stacking structure that has spectroscopic triplex characteristics. The CD spectra of (CUG)5 RNA and an isobasic randomized control at 20 °C are shown in Fig. 2 B. The (CUG)5 spectrum has a positive band at 269 nm, a small negative band at 237 nm, and a large negative band at 208 nm. The 208 nm band, which is indicative of RNA base pairing and stacking (38Gray D. Liu J. Ratcliff R.L. Allen F.S. Biopolymers. 1981; 20: 1337-1382Crossref Scopus (91) Google Scholar), is not present in the randomized control, suggesting that (CUG)5 RNA is significantly more base-paired and stacked than the control. The long wavelength peak (at 269 nm rather than the 285 nm of the corresponding DNA and 282 nm for the RNA control) is very sensitive to base composition and RNA conformation (38Gray D. Liu J. Ratcliff R.L. Allen F.S. Biopolymers. 1981; 20: 1337-1382Crossref Scopus (91) Google Scholar, 39Causley G.C. Johnson W.C.J. Biopolymers. 1982; 21: 1763-1780Crossref PubMed Scopus (30) Google Scholar). The overall spectrum is very similar to those obtained for RNAs containing pseudoknots with a small but significant negative band at ∼237 nm (40Johnson K.H. Gray D.M. J. Biomol. Struct. Dyn. 1992; 9: 733-746Crossref PubMed Scopus (14) Google Scholar, 41Shi P.-Y. Brinton M.A. Veal J.M. Zhong Y.Y. Wilson W.D. Biochemistry. 1996; 35: 4222-4230Crossref PubMed Scopus (113) Google Scholar) (naturally occurring A-form RNAs generally lack any marked band at this wavelength (42Johnson K.H. Gray D.M. Biopolymers. 1991; 31: 385-395Crossref PubMed Scopus (9) Google Scholar, 43Loret E.P. Georgel P. Johnson W.C.J. Ho P.S. Proc. Natl. Acad. Aci. U. S. A. 1992; 89: 9734-9738Crossref PubMed Scopus (78) Google Scholar, 44Newbury S.F. McClellan J.A. Rodger A. Anal. Commun. 1996; 33: 117-121Crossref Scopus (5) Google Scholar)). The spectrum of the intermolecular triplex formed by poly(A)-poly(G)-poly(C) is also similar to that of the (CUG) repeat, except that the small negative band for this intermolecular triplex is at 245 nm (33Chastain M. Tinoco I.J. Nucleic Acids Res. 1992; 20: 315-318Crossref PubMed Scopus (21) Google Scholar). Since an intramolecular pseudoknot might be described as a pseudo-triplex, these two descriptions are equally valid. To analyze the effect of temperature on (CUG)5 RNA, we measured the CD spectra at a range of temperatures between 5 and 80 °C (Fig. 2 C). The negative band at 208 nm decreases markedly with increased temperature as the base pairs melt apart (44Newbury S.F. McClellan J.A. Rodger A. Anal. Commun. 1996; 33: 117-121Crossref Scopus (5) Google Scholar). At 65 °C, the 208 nm band is absent and the 267 nm band decreases and shifts to 275 nm resembling the randomized control, indicating that the (CUG)5 structure is completely melted. The series of spectra obtained are almost identical to CD spectra of a 59-nucleotide flavivirus pseudoknot at various temperatures (41Shi P.-Y. Brinton M.A. Veal J.M. Zhong Y.Y. Wilson W.D. Biochemistry. 1996; 35: 4222-4230Crossref PubMed Scopus (113) Google Scholar). The melting curve for (CUG)5 (Fig. 2 D) was analyzed following (25Marky L.A. Breslauer K.J. Biopolymers. 1987; 26: 1601-1620Crossref PubMed Scopus (1097) Google Scholar) to give a single transition with melting temperature of 54.8 °C. The van't Hoff transition enthalpy of the transition was determined from Fig. 2 F(T max = 328.1 and T 2 = 336.6 K) to be ΔH VH = (190 ± 30) kJ mol−1 and ΔS = (580 ± 90) J K−1 mol−1. A single transition is not inconsistent with the possibility that the structure may resemble a pseudoknot; detailed studies of the thermal melting of pseudoknots show that they can melt with one apparent transition (45Spedding G. Gluik T.C. Draper D.E. J. Mol. Biol. 1992; 229: 609-622Crossref Scopus (35) Google Scholar, 46Gluick T. Draper D. J. Mol. Biol. 1994; 241: 246-262Crossref PubMed Scopus (87) Google Scholar). However,T m and ΔH values are high for a 15-mer of any kind, especially as this usually means <8 base pairs. For example, the T m for a duplex with a similar ratio of G/C and A/U in 1 m NaCl (GUCUAGAC) (versus 5 mm sodium phosphate in our experiments) is 56.2 °C (47Freier S.M. Kierzek R. Jaeger J.A. Sugimoto N. Caruthers M.H. Neilson T. Turner D.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9373-9377Crossref PubMed Scopus (1255) Google Scholar) and the T m and ΔH values for a 23-mer RNA hairpin (with 7 Watson-Crick base pairs) in 10 mmsodium phosphate are 50.9 °C and 193 kJ mol−1, respectively (48Sarkar M. Sigurdsson S. Tomac S. Sen S. Rozners E. Sjoberg B.-M. Stromberg R. Graslund A. Biochemistry. 1996; 35: 4678-4688Crossref PubMed Scopus (22) Google Scholar). Thus whatever structure is adopted is very stable and is unlikely to be a hairpin. The RNA containing (CUG)50 by way of contrast has two thermal transitions occurring at ∼40 °C and 76 °C (Fig.2 H) and a CD spectrum resembling that of A-form RNA (Fig.2 G). It is interesting to note that there are two bands when this sequence is run on a non-denaturing gel (Fig. 1 F). It may therefore be that the two transitions reflect two distinct species melting rather than one species undergoing a two stage transition. A conformational study of a single strand of RNA was undertaken to assess the viability of the proposed structure (see below). This was not intended to be a definitive modeling study, indeed the scope of this study would not allow for such a major computational undertaking, but rather to ensure that the proposed structure was reasonable; in particular it should be thermally accessible and represent at least a local free energy minimum structure. Calculations were performed o
Referência(s)