Anu Sheth

Solution structure of d-GAATTCGAATTC by 2D NMR: A new approach to determination of sugar geometries in DNA segments

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d‐GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare Ol′‐endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2′‐endo and C4′‐endo.

Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Two dimensional (2D) FT-NMR investigations have been carried out on the self-complementary dodecanucleotide d-CTCGAGCTCGAG, which has cleavage sites for the restriction enzyme Xho I (between C and T). The central TCG portion is also known to show a preference for DNAase activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the COSY and NOESY spectra. The results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central TCG portion also exhibits variations in the backbone structure. The base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches.

Analysis of resolution and sensitivity enhancements in two-dimensional scaling experiments

General criteria have been described for evaluating resolution and sensitivity enhancements in two-dimensional scaling experiments. Three different scaling schemes have been considered and experimental results showing a comparison of these are presented. Finally, optimization of the scaling experiments has been discussed.

Application of J scaling in two-dimensional spin-echo-correlated spectroscopy to observation of small coupling correlations

Abstract The idea of J scaling along the ω 1 axis in two-dimensional correlated spectroscopy has been extended to spin-echo-correlated spectroscopy (SECSY). A detailed theoretical analysis of the pulse scheme and the extent of sensitivity enhancement as a result of J scaling has been given. The technique is shown to be particularly suitable for observation of small coupling correlations since it avoids the use of refocusing delays prior to data aquisition. Experimental spectra showing small coupling correlations of the order of 0.5 Hz, in J -scaled SECSY are presented.

A novel loop structure observed in d-GAATTCCCGAATTC by 2D NMR

Summary Sequential resonance assignments of the non exchangeable base and sugar protons in d-GAATTCCCGAATTC have been obtained using two dimensional NMR experiments at 500 MHz. The chemical shifts and the NOEs have been used to determine the structure in the base-pair mismatch region which is located in the central portion of the molecule. It is observed that the molecule adopts a novel unsymmetrical loop structure in this section which is characterised by sugar geometries which are significantly different compared to the rest of the molecule. The base-paired portion of the molecule conforms to a right handed B-DNA type of structure.

Refined procedures for accurate determination of solution structures of nucleic acids by two dimensional nuclear magnetic resonance spectroscopy

New procedures have been described for accurate determination of solution structures of nucleic acids. These are two fold; new two dimensional nuclear magnetic resonance techniques and better approaches for interpretation of nuclear magnetic resonance data for structure determination purposes. The significant development in two dimensional nuclear magnetic resonance techniques for this purpose areω1 -scaling and recording of pure phase spectra. Use ofω1-scaled correlated and nuclear Overhauser effect spectra for estimation of interproton distances and 1H-1H coupling constants has been described. Computer simulation procedures for exact determination of structure have been described. Experimental spectra demonstrating the application of new procedures have been presented.

Creation of zero-quantum coherences from equilibrium spin systems

Abstract It is demonstrated that zero-quantum coherence can be created by a single non-selective, pulse from equilibrium spin systems which are isotropically coupled. The zero-quantum coherence essentially arises from the mixing of states. A two-dimensional NMR pulse scheme is designed to observe the zero-quantum cross peaks in systems with small scalar coupling constants by exploiting the relatively long transverse relaxation times of the zero-quantum coherence.

Combined use of COSS and S.COSY for recording of pure-phase shift-scaled spectra☆

The phase characteristics of two chemical-shift scaling pulse sequences COSS and S.COSY have been analyzed, and a method for eliminating dispersive contributions to cross and diagonal peaks in the 2D spectra has been suggested. This results in pure absorption peak shapes for both diagonal- and cross-peak components and both acquire in-phase characteristics. An experimental demonstration of the procedure is given with a one-dimensional simplification due to practical limitations of the available 2D software.

Computer simulation of the cross peak patterns in noesy spectra of oligonucleotides: an aid to accurate structure determination in aqueous solution

Computer simulation of two dimensional NOESY spectra of oligonucleotides is described as an aid to structure determination of nucleic acids in solution. It is shown that the patterns of cross peaks help identification of DNA-structural family by mere inspection. Illustrative examples are shown with known assignments in d-CTCGAGCTCGAG.