Restricting the Conformational Heterogeneity of RNA by Specific Incorporation of 8-Bromoguanosine David J. Proctor, ² Elzbieta Kierzek, ‡ Ryszard Kierzek,* ,‡ and Philip C. Bevilacqua* ,² Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802, and Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-704 Poznan, Noskowskiego 12/14, Poland Received October 31, 2002 ; E-mail: pcb@chem.psu.edu; rkierzek@rose.man.poznan.pl RNA typically folds in a hierarchical fashion, forming indepen- dently stable secondary structure before tertiary structure. 1 The function of many RNAs depends on a compact tertiary structure, as exemplified by a number of small ribozymes. 2 Unfortunately, secondary structure is prone to alternative pairings, or misfolds, which hinder the formation of native tertiary structure. 3 Because these interactions are strong, misfolds can lead to kinetic trapping, complicating mechanistic and structural studies of RNA. Consider- able effort has been put into correcting misfolds to produce fast- folding RNAs. Since misfolding occurs most frequently at the secondary structural level, site-directed mutagenesis and antisense oligonucleotides have provided simple approaches to promote native folding, 3 as have nucleotide analogues. 4 Additionally, proteins have been shown to facilitate RNA folding in vitro and in vivo, 5 and variation in pH, temperature, metal ion, and RNA concentration can reduce RNA conformational heterogeneity. 3 One example of secondary structural misfolding is the dimerization of hairpins to give a duplex with a symmetric internal loop (Figure 1A). Dimerization is especially problematic at high RNA and salt concentrations, such as those required for NMR and X-ray crystallographic studies. 6 Even the unusually stable UUCG tetraloop hairpin can form a duplex during crystallization. 7 Here, we describe limiting the conformational heterogeneity of RNA using the nucleotide analogue 8-bromoguanosine (8BrG). Structural studies on nucleosides and polymers have shown that 8BrG preferentially adopts the syn conformation, wherein the nucleobase is positioned over the ribose sugar. 8 This conformation, which is in contrast to the anti conformation typical of A-form RNA helices, arises because the steric bulk of bromine precludes its residence over the ribose ring. We demonstrate that 8BrG shifts a hairpin-duplex equilibrium toward the hairpin conformation primarily by destabilizing the duplex conformation (Figure 1). The 8BrG analogue was introduced into loop position 4 of selected YNMG hairpin tetraloops (Figure 1A). The YNMG motif is comprised of 16 thermodynamically stable sequences that adopt structures similar to the UUCG tetraloop. 6 The YNMG motif was chosen as a model system since a syn guanosine occurs naturally at position 4 of the loop, and inspection of YNMG structures suggests substitution should not result in a significant steric clash. In addition, the YNMG motif gives rise to several diagnostic NMR spectroscopic features, including an unusually upfield-shifted imino proton resonance that is due to a bifurcated hydrogen bond between positions 1 (Y ) C or U) and 4 (G) of the loop, and an unusually downfield-shifted 31 P resonance caused by a sharp turn in the backbone at G9. 6,9 8-bromoguanosine was synthesized as a phosphoramidite and incorporated into 12mer RNA oligonucleotides (Table 1) using standard chemistry. 10 Thermodynamic characterization (Table 1) revealed that substitution at position 4 of the UUCG loop, UUCG, had little effect upon stability, with ΔΔG° 37 )-0.08 ( 0.21 kcal mol -1 and ∆T M ) 1.2 °C relative to UUCG. Likewise, 8BrG- substitution at position 4 of the CGCG loop, CGCG, did not have a significant effect, with ∆∆G° 37 )-0.41 ( 0.58 kcal mol -1 and ∆T M ) 1.5 °C relative to CGCG. Structural characterization of unmodified UUCG by 1D 1 H- decoupled 31 P NMR spectroscopy revealed the expected 11 hairpin resonances dispersed over ∼2 ppm (Figure 2A). These include a resonance downfield-shifted to 1.15 ppm, which was previously assigned to G9P. 9 As expected on the basis of similar thermody- namic parameters (Table 1), the spectrum of 8BrG-substituted UUCG was nearly identical to UUCG (Figure 2B). In contrast to UUCG, the 31 P spectrum for CGCG comprised several densely packed resonances covering only ∼1 ppm (Figure 2C). One-dimensional 1 H-decoupled 31 P NMR spectra of A-form RNA helices have chemical shifts clustered near 0 ppm (referenced ² The Pennsylvania State University. ‡ Polish Academy of Sciences. Figure 1. (A) Equilibrium between hairpin and duplex conformations. A red G indicates 8BrG substitution; in the text this is indicated by a bold and underlined G.Y ) C or U; N ) A, C, G, or U; M ) A or C. (B) Free energy diagram depicting the destabilization of the duplex conformation upon 8BrG substitution; h ) hairpin; d ) duplex; u ) unfolded. Table 1. Thermodynamic Parameters for Hairpin Formation 10 sequence a ∆G° 37 (kcal mol -1 ) b TM (°C) c ggacUUCGgucc -4.80 ( 0.13 71.7 gGacUUCGgucc -2.44 ( 0.12 57.2 ggacUUCGgucc -4.88 ( 0.17 72.9 ggacCGCGgucc d -3.60 ( 0.36 67.0 ggacCGCGgucc -4.01 ( 0.45 68.5 a The hairpin tetraloop is capitalized. b An extra significant figure is shown for ∆G° 37 to avoid round-off errors. c Maximum errors in TM are ∼1 °C. d Collected at 5-50 μM to favor the hairpin conformation. Figure 2. 1 H-decoupled 31 P NMR spectra (202 MHz, 95% H2O/5% D2O in 10 mM NaH2PO4/0.1 mM EDTA, pH 7.0, 45 °C) of (A) 0.4 mM UUCG, (B) 0.3 mM UUCG, (C) 0.4 mM CGCG, (D) 0.2 mM CGCG; and with 1M Na + , (E) 0.2 mM CGAG, and (F) 0.5 mM CGAG. 13 The dominant conformation is given, and the downfield-shifted resonance diagnostic of the hairpin is indicated with a filled dot. Published on Web 02/11/2003 2390 9 J. AM. CHEM. SOC. 2003, 125, 2390-2391 10.1021/ja029176m CCC: $25.00 © 2003 American Chemical Society