Imino Proton NMR Research Articles

Human Telomere Quadruplex: Refolding and Selection of Individual Conformers via RNA/DNA Chimeric Editing

The conformation of the guanine quadruplex formed by the human telomere (HT) repeat in solutions containing physiological concentrations of K+ ions has been a topic of intensive investigation during the past several years. Of particular interest are the directionality of the overall folding pattern, i.e., parallel, antiparallel, or a combination of these two modes, and the alternation, if any, of the glycosidic bond conformation between syn and anti. An additional issue involves resolving mixtures of conformations when more than one species is present. We approach these questions using selective substitution of riboguanosine, rG, for deoxyriboguanosine, dG. Using a combination of circular dichroism, gel electrophoresis, equilibrium ultracentrifugation, and imino proton NMR, we are able to show that these modifications can yield sequences which fold into parallel or antiparallel conformations consisting of one or two strands. We also demonstrate that chimeric editing of the HT sequence permits isolating one of two conformational isomers existing in solution in the presence of KCl. The ability to engineer and control quadruplex folding motifs illustrated here with HT may prove useful more generally for a variety of quadruplex-forming sequences.

The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs.

Uracil DNA glycosylase (UNG) locates uracil and its structural congener thymine in the context of duplex DNA using a base flipping mechanism. NMR imino proton exchange measurements were performed on free and UNG-bound DNA duplexes in which a single thymine (T) was paired with a series of adenine analogues (X) capable of forming one, two, or three hydrogen bonds. The base pair opening equilibrium for the free DNA increased 55-fold as the number of hydrogen bonds decreased, but the opening rate constants were nearly the same in the absence and presence of UNG. In contrast, UNG was found to slow the base pair closing rate constants (kcl) compared to each free duplex by a factor of 3- to 23-fold. These findings indicate that regardless of the inherent thermodynamic stability of the TX pair, UNG does not alter the spontaneous opening rate. Instead, the enzyme holds the spontaneously expelled thymine (or uracil) in a transient extrahelical sieving site where it may partition forward into the enzyme active site (uracil) or back into the DNA base stack (thymine).

NMR imino proton exchange experiments on duplex DNA primarily monitor the opening of purine bases.

Molecular dynamics simulations were performed to investigate GC and AT base opening events in DNA. Calculated equilibrium constants between the base open (or flipped) and closed states were shown to be in good agreement with experimental data from NMR imino proton exchange experiments. Analysis of the computed results indicates that the equilbrium constants are dominated by the opening of the A and G bases in the AT and GC base pairs, respectively. Thus, the present results predict that NMR imino proton exchange experiments of base opening are primarily monitoring the opening of purine bases.

Synthesis and redox-active base-pairing properties of DNA incorporating mercapto C-nucleosides

Here, we describe the synthesis and incorporation of the nucleoside base analogue C-deoxyribonucleoside 3 carrying thiophenol into DNA. The 1′-β compound 3 was synthesized by Friedel–Crafts alkylation, followed by deprotection. The coupling reaction with 3,5-ditoluoyl-1-α/β-methoxy-2-deoxy- d-ribose and diphenyldisulfide in the presence of SnCl 4 afforded the α/β mixture 2 (β/α=2.8), and the β-form was separated by silica gel chromatography. After formation of the phosphoramidite derivative, the C-nucleoside 3 was incorporated into DNA. When the mercapto-bases were incorporated into complementary singled-stranded (ss) DNAs, the resulting duplex displayed high thermal stabilization on treatment with bubbling O 2 ( T m 73 °C), but was destabilized in the presence of mercaptoethanol ( T m 33 °C). CD spectra showed that the duplex had a right-handed double-stranded structure. Imino proton NMR studies of temperature stability suggested that the strength of hydrogen bonding around the mercapto C-nucleoside was larger when treated with bubbling O 2 than when in treated with reducing agent. Thus, formation of the base-to-base disulfide bond increased the stability of the duplex; correspondingly, reduction of the disulfide to two thiol bases destabilized the DNA reversibly. The duplex-forming disulfide base pair showed resistance to exonulease III . The present strategy could be used to introduce new functionalities into cells and novel biomaterials.

Dynamic opening of DNA during the enzymatic search for a damaged base

Uracil DNA glycosylase (UDG) removes uracil from U.A or U.G base pairs in genomic DNA by extruding the aberrant uracil from the DNA base stack. A question in enzymatic DNA repair is whether UDG and related glycosylases also use an extrahelical recognition mechanism to inspect the integrity of undamaged base pairs. Using NMR imino proton exchange measurements we find that UDG substantially increases the equilibrium constant for opening of T-A base pairs by almost two orders of magnitude relative to free B-DNA. This increase is brought about by enzymatic stabilization of an open state of the base pair without increasing the rate constant for spontaneous base pair opening. These findings indicate a passive search mechanism in which UDG uses the spontaneous opening dynamics of DNA to inspect normal base pairs in a rapid genome-wide search for uracil in DNA.

Factors affecting thermodynamic stabilities of RNA 3 x 3 internal loops.

Internal loops in RNA are important for folding and function. The 3 x 3 nucleotide internal loops are the smallest size symmetric loops with a potential noncanonical base pair (middle pair) flanked on both sides by a noncanonical base pair (loop-terminal pair). Thermodynamic and structural insights acquired for 3 x 3 loops should improve approximations for stabilities of 3 x 3 and larger internal loops. Most natural 3 x 3 internal loops are purine rich, which is also true of other internal loops. A series of oligoribonucleotides containing different 3 x 3 internal loops were studied by UV melting and imino proton NMR. Both loop-terminal and middle pairs contribute to the thermodynamic stabilities of 3 x 3 loops. Extra stabilization of -1.2 kcal/mol was found for a GA middle pair when flanked by at least one non-pyrimidine-pyrimidine loop-terminal pair. A penalty of approximately 1 kcal/mol was found for loops with a single loop-terminal GA pair that has a U 3' to the G of the GA pair. A revised model for predicting stabilities of 3 x 3 loops is derived by multiple linear regression.

Bistable Secondary Structures of Small RNAs and Their Structural Probing by Comparative Imino Proton NMR Spectroscopy

We investigate 25–34 nucleotide RNA sequences, that have been rationally designed to adopt two different secondary structures that are in thermodynamic equilibrium. Experimental evidence for the co-existence of the two conformers results from the NH⋯N 1H NMR spectra. When compared to the NH⋯N 1H NMR spectra of appropriate reference sequences the equilibrium position is easily quantifiable even without the assignment of the individual NH resonances. The reference sequences represent several Watson–Crick base-paired double helical segments, each encountered in either of the two conformers of the bistable target sequence. In addition, we rationalize the influence of nucleotide mutations on the equilibrium position of one of the bistable RNA sequences. The approach further allows a detailed thermodynamic analysis and the evaluation of secondary structure predictions for multistable RNAs obtained by computational methods.

Synthesis of helix 69 of Escherichia coli 23S rRNA containing its natural modified nucleosides, m(3)Psi and Psi.

The synthesis of 3-methylpseudouridine (m(3)Psi) phosphoramidite, 5'-O-[benzhydryloxybis(trimethylsilyloxy)silyl]-2'-O-[bis(2-acetoxyethoxy)methyl]-3-methylpseudouridine-3'-(methyl-N,N-diisopropyl)phosphoramidite, is reported. Selective pivaloyloxymethyl protection of the Psi N1 followed by methylation at N3 was used to generate the naturally occurring pseudouridine analogue. The m(3)Psi phosphoramidite was used in combination with pseudouridine (Psi) and standard base phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S ribosomal RNA (rRNA) (residues 1906-1924), containing a single m(3)Psi at position 1915 and two Psi's at positions 1911 and 1917. Our synthesis of the fully modified helix 69 RNA demonstrates the ability to make milligram quantities of RNA that can be used for further high-resolution structure studies. Site-selective introduction of the methyl group at the N3 position of pseudouridine at position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar stability. One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.

Imino proton NMR analysis of HDV ribozymes: nested double pseudoknot structure and Mg2+ ion-binding site close to the catalytic core in solution.

Minimized trans-acting HDV ribozyme systems consisting of three (Rz-3) and two (Rz-2) RNA strands were prepared and their folding conformations were analyzed by NMR spectroscopy. The guanosine residues in one of the enzyme components of Rz-3 were labeled with 13C and 15N. Imino proton signals were assigned by analysis of NOESY and HSQC spectra. The results are consistent with the nested double pseudoknot model, which contains novel base pairs (P1.1), as observed in the crystal structure of a genomic HDV ribozyme. The NOE connectivities suggest an additional G:G pair at the bottom of P1.1 and at the top of P4. The effects of temperature and Mg2+ ions on base pairs for Rz-3 were examined. The temperature variation experiment on Rz-3 showed that P3 is the most stable and that P1.1 is as stable as P1 and P2. The imino proton signals of the G:U pair at the bottom of P1 and the top of P1.1, which are close to the cleavage site, showed the largest changes upon Mg2+ titration of Rz-3. The results suggest that the catalytic Mg2+ ion binds to the pocket formed by P1 and L3.

Thermodynamic stabilities of internal loops with GU closing pairs in RNA.

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.

Thermodynamics of RNA internal loops with a guanosine-guanosine pair adjacent to another noncanonical pair.

Thermodynamic parameters measured by optical melting are reported for formation of RNA duplexes containing tandem noncanonical pairs with at least one guanosine-guanosine (GG) pair. For selected sequences, imino proton NMR provides evidence that the desired duplex forms and that the structure of a GG pair adjacent to a noncanonical pair depends on context. A GG pair next to a different noncanonical pair is more stable than expected from measurements of adjacent GG pairs. This is likely due to an unfavorable stacking interaction between adjacent GG pairs, where areas of high negative charge probably overlap. The results suggest a model where tandem noncanonical pairs closed by two GC pairs are assigned the following free energy increments at 37 degrees C: 0.8 kcal/mol for adjacent GG pairs, 1.0 kcal/mol for GG next to UU, and -0.3 kcal/mol for all others. These values are adjusted by 0.65 kcal/mol for each closing AU pair.

Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA.

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.

Intramolecular Quadruplex Formation of the G-Rich Strand of the Mouse Hypervariable Minisatellite Pc-1

The minisatellite Pc-1, isolated from the mouse genome consisting of a tandem repeat of d(GGCAG), is hypervariable with a mutation rate of 0.15/generation. Here we describe a structural characterization of the G-rich strand of Pc-1 by biochemical and physicochemical methods. It was found to be comparatively resistant to both single-stranded DNA-binding protein binding and digestion by single-stranded DNA-specific nuclease and to cause arrest of DNA synthesis. The guanine imino proton NMR signals observed on the Pc-1 G-rich strand and their slow 1H/2H exchange profiles pointed to a quadruplex structure with guanine quartets. The melting temperature of the quadruplex determined by CD was not dependent on DNA concentration. These results indicate that the G-rich strand of Pc-1 forms an intramolecular folded-back quadruplex structure under physiological conditions. Possible mechanisms of the Pc-1 mutations implicated with the formation of the quadruplex structure are discussed.

NMR spectroscopic and enzymatic studies of DNA hairpins containing mismatches in the EcoRI recognition site

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ·mol-1 less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.Key words: DNA hairpins, EcoRI recognition, mismatches, imino protons.

NMR spectroscopic and enzymatic studies of DNA hairpins containing mismatches in the EcoRI recognition site.

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ x mol(-1) less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.

Thermodynamics of nonsymmetric tandem mismatches adjacent to G.C base pairs in RNA.

The thermodynamic stabilities and structures of a series of RNA duplexes containing nonsymmetric tandem mismatches in the context of , where are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in determining loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.

Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability.

In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].

Thermodynamics of folding of the RNA pseudoknot of the T4 gene 32 autoregulatory messenger RNA.

Nucleotides U(-67) to C(-40) at the extreme 5' end of the gene 32 mRNA in bacteriophage T4 have been shown to fold into an RNA pseudoknot proposed to be important for translational autoregulation. The thermal denaturation of three in vitro transcribed RNAs corresponding to the pseudoknot region has been investigated as a function of Mg2+ concentration to begin to elucidate the determinants of the structure and stability of this conformation. T4-35 is a 35-nucleotide RNA containing a 5' G followed by the natural T4 sequence starting with the mature 5' end of the mRNA, nucleotides A(-71) to C(-38). A 32-nucleotide RNA, termed T4-32, contains the native sequence form U(-67) to C(40) with 5'GC and 5'CA single-stranded regions appended to the 5' and 3' ends of the core sequence, respectively. T4-28 contains only the 28 core nucleotides, and the predicted closing U(-67)-A(-52) base pair in stem 1 has been replaced with a phylogenetically allowed G(-67)-C(-52) base pair. Ribonuclease mapping of T4-32 and imino proton NMR experiments of T4-35 show that both sequences adopt a pseudoknotted conformation. At pH 6.9 and 50 mM NaCl, T4-35 and T4-32 RNAs are characterized by a single major melting transition over a wide range of [Mg2+] (0-6 mM). The delta H degree of unfolding for T4-35 and T4-32 shows a large dependence on Mg2+ concentration; the maximum delta H degree occurs at about 2.0 mM Mg2+ with further addition of Mg2+ simply increasing the tm. Investigation of the [Mg2+] dependence of the tm suggests that a net of one Mg2+ ion is released upon denaturation of T4-35 and T4-32 RNAs. Over the entire [Mg2+] range, the delta G degree (37 degrees C) for the folding of T4-35 is consistently 1-1.5 kcal mol(-1) more negative than T4-32 due to a higher stabilization enthalpy for the natural sequence molecule. In contrast to this behavior, T4-28 gives consistently higher tm's but less negative enthalpies and is destabilized (at 37 degrees C) by about 0.5-1.5 kcal mol(-1) relative to T4-32 and by about 2-3 kcal mol(-1) relative to T4-35, depending upon cation concentration. (1)H NMR experiments suggest that, even in the presence of 4.0 mM Mg2+, T4-28 RNA does not adopt a stable pseudoknotted conformation. These data show that the stability of the pseudoknot in the gene 32 mRNA encoded by the 28-nucleotide core sequence is significantly influenced by the number and nature of the immediately adjacent "single-stranded" 5' and/or 3' nucleotides appended to the core structure. These findings are discussed within the context of the structural model for the evolutionarily related phage T2 and T6 gene 32 mRNA pseudoknots presented in the following paper [Du, Z., Giedroc, D. P., & Hoffman, D. W. (1996) Biochemistry 35, 4187-4198].

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

A periodic table of symmetric tandem mismatches in RNA.

The stabilities and structures of a series of RNA octamers containing symmetric tandem mismatches were studied by UV melting and imino proton NMR. The free energy increments for tandem mismatch formation are found to depend upon both mismatch sequence and adjacent base pairs. The observed sequence dependence of tandem mismatch stability is UGGU > GUUG > GAAG > or = AGGA > UUUU > CAAC > or = CUUC approximately UCCU approximately CCCC approximately ACCA approximately AAAA, and the closing base pair dependence is 5'G3'C > 5'C3'G > 5'U3'A approximately 5'A3'U. These results differ from expectations based on models used in RNA folding algorithms and from the sequence dependence observed for folding of RNA hairpins. Imino proton NMR results indicate the sequence dependence is partially due to hydrogen bonding within mismatches.