Maurice Guéron

The i-motif in nucleic acids.

Seven years after the discovery of the DNA i-motif, partial explanations for its occurrence have been uncovered, possibly involving CHellipsisO hydrogen bonds across the narrow grooves. Investigations of its biological significance have been encouraged by the demonstration and description of the intramolecular i-motif structure of human telomeric and centromeric sequences, by the recent observation of an intercalated RNA structure and by the discovery of proteins that associate with DNA sequences carrying cytosine repeats. The compatibility of the intercalation with peptide and phosphorothioate DNA analogs is favorable for possible pharmaceutical applications.

[16] Studies of base pair kinetics by NMR measurement of proton exchange

Publisher Summary This chapter discusses the studies of base pair kinetics by nuclear magnetic resonance (NMR) measurement of proton exchange. Base pairing is ubiquitous in nucleic acids; it plays a key role in the structure and function of biological molecules (B-DNA, tRNA, etc.) and of biochemical constructs in PCR, antisense strategy, etc. The systematic disruption of base pairs is a prerequisite for replication and transcription of double-stranded DNA. Local disruption could play a role in mechanical properties of nucleic acids and in the specificity of protein-nucleic acid recognition. It is involved in chemical reactions of nucleic acids. As a probe of structural properties, the kinetics of base pair opening forms a useful complement to structure determination by NMR. The most common approach to base pair opening is the measurement of imino-proton exchange, with water, by proton NMR. With NMR, an exchange event can be directly assigned to an imino (rather than amino or ribose) proton of a specific base pair, in contrast to the earlier procedures based on isotope exchange, monitored by radioactivity or ultraviolet, infrared, or Raman spectroscopy. The measurement methods are of two types: the kinetics of real-time exchange and of magnetization transfer measure exchange times directly, whereas the measurements of longitudinal relaxation and line broadening can only determine the differential effect of added catalyst on the exchange time.

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton☆

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.

Sensitive CEST agents based on nucleic acid imino proton exchange: detection of poly(rU) and of a dendrimer-poly(rU) model for nucleic acid delivery and pharmacology.

It is shown that the exchange properties of the imino and hydroxyl protons of polyuridilic acid (poly(rU)) allow use of this compound as a chemical‐exchange saturation transfer (CEST) contrast agent. A proton/proton sensitivity enhancement factor of over 5000 per imino proton allowed the detection of a few micromolar of polymer (2000 uridine units; 644 kD) with a 50% change in the water signal. The enhancement factor would increase further at even lower concentrations, opening up the submicromolar range. When poly(rU) was complexed to a dendrimer carrying 250 positive charges, the stoichiometry was approximately one RNA for 10 dendrimers. The sensitivity enhancement was reduced but remained large (2300/imino proton), bringing enhanced CEST visibility to the dendrimers. The net charge of the complex was positive, suggesting that the complexed dendrimers would still interact with cell membranes, and that the RNA‐dendrimer complex could provide a model for a gene delivery system with good CEST visibility. Magn Reson Med 49:998–1005, 2003.

Solution structures of the i-motif tetramers of d(TCC), d(5methylCCT) and d(T5methylCC): novel NOE connections between amino protons and sugar protons.

BACKGROUND At slightly acid or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines form a tetramer structure in which two parallel-stranded duplexes have their hemi-protonated C.C+ base pairs face-to-face and fully intercalated, in a so-called i-motif, first observed serendipitously in [d(TC5)]4. RESULTS A high-definition structure of [d(TCC)]4 was computed on the basis of inter-residue distances corresponding to 21 NOESY cross-peaks measured at short mixing times. A similarly defined structure of [d(5mCCT)]4 was also obtained. A small number of very characteristic (amino proton)-(sugar proton) cross-peaks entails the intercalation topology. The structure is generally similar to that of [d(TC5)]4. The sequence d(T5mCC) forms two tetramers in comparable proportions. The intercalation topologies are read off the two patterns of (amino proton)-(sugar proton) cross-peaks: one is the same as in the d(TCC) tetramer, the other has the intercalated strands shifted by one base, which avoids the steric hindrance between the methyl groups of the 5mC pairs of the two duplexes. CONCLUSIONS The structures obtained in this work and the procedures introduced to characterize them and to solve the problems linked to the symmetry of the structure provide tools for further exploring the conditions required for formation of the i-motif.

Solvent-peak-suppressed NMR: Correction of baseline distortions and use of strong-pulse excitation

Abstract Pulse excitation sequences which involve time delays lead to spectra suffering from so-called frequency-dependent phase shifts. The phenomenon is analyzed in a model case, and the above description is shown to be somewhat improper. Rather, the spectrum should be described as a sum of Lorentzian lines, each of which has its own, frequency-independent , phase shift. The analysis leads to procedures for correcting the baseline distortions of solvent-suppressed NMR based on the Redfield 2-1-4 sequence, or on analogous strong-pulse sequences which are described. Goals and methods of solvent-signal suppression are discussed in this context.

The inhibition of bovine heart hexokinase by 2-deoxy-d-glucose-6-phosphate: characterization by 31P NMR and metabolic implications

The glucose analog, 2-deoxy-D-glucose (2DG), has been used widely for studying the initial steps in the metabolism of glucose by radio-isotope tracer methods and by 31P NMR. In the rat heart perfused with acetate/2DG (both 5 mM) plus insulin, trapping of phosphorus by 2-deoxy-D-glucose-6-phosphate (2DG6P) results in a steady state exhibiting high 2DG6P (55 mM) and low ATP concentrations but near-normal function, as observed in an earlier 31P NMR study. In order to understand how the 2DG6P concentration is stabilized, we studied the inhibition of a mammalian hexokinase by 2DG6P in vitro by a 31P NMR technique. Inhibition, previously unobserved, was found. It is similar to inhibition by G6P in that it is competitive with ATP and not competitive with 2DG, but the inhibition constant (1.4 mM) is much larger. The experimental protocol includes provisions for enzymatic destruction of stray inhibitors such as G6P. The results show that the high 2DG6P and low ATP concentrations found in the steady state of the perfused heart should strongly reduce the rate of phosphorylation of sugars by hexokinase.

Proton Magnetic Relaxation Study of the Manganese–Transfer‐RNA Complex

At 20°C, transfer ribonucleic acid (tRNA) binds manganese ions cooperatively. The binding sites are studied by spin relaxation of the water protons in a solution of the Mn2+–tRNA complex, as a function of temperature and proton NMR frequency. Below room temperature the frequency dependence of the proton relaxation shows that the correlation time τc of the ion–proton dipolar interaction is limited by electron spin relaxation. Hence τc is not in these conditions an indicator of molecular motion. This explains why the relaxation effects of Mn2+ bound to different polynucleotides were previously observed to be quite similar. This also shows that the rotational correlation time is long (> 2 × 10−9 sec) and hence there is rigidity in the binding of the ion to tRNA. The “effective” number, pw, of water molecules in the first hydration shell, taking into account possible anisotropies of the molecular movements, is equal to two. This may indicate that the first hydration shell contains only two water molecules, th...

Internal motions of transfer RNA: A study of exchanging protons by magnetic resonance

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H2O samples in D2O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CG11 of yeast tRNAPhe at 17 degrees C, or 5 minutes for U8-A14 of yeast tRNA(Asp) at 20 degrees C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other, fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA(Phe) has a lifetime of 14 ms at 17 degrees C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether short-lived or long-lived. Buffer effects have also been observed in poly(rA).poly(rU), for which we derive a lifetime of 2.5 ms at 27 degrees C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.

Line narrowing and line broadening using trigonometric functions

The sine-bell narrowing routine (De Marco and Wuthrich, J. Magn. Resonance24, 201 (1976)) yields rigorously a finite differential of the dispersion spectrum The convolution difference routine operates in a similar fashion. Sine multiplication is generalized. We propose standard routines for (a) line narrowing, (b) line narrowing and shaping, (c) line broadening.