H. Todd Miles

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

The use of new 1H‐detected heteronuclear 1H‐31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4′ and C5′ sugar protons. Use of the new methods enables one to make sequence‐specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.

Helix formation between polyribonucleotides and purines, purine nucleosides and nucleotides. II

A number of monomeric nucleic acid components react with polynucleotides to form well-defined complexes having helical structures similar to those formed between corresponding pairs of high polymers. The monomer can be a purine or a purine nucleoside or nucleotide, but no pyrimidine derivative has yet been found to form such complexes. The phenomenon has been investigated by infrared spectroscopy and optical rotation and by use of an enzyme of known sensitivity to secondary structure. Base-pairing specificity of interaction is observed, and the components react in definite stoichiometric ratios. The dependence of the stabilities of some of the complexes upon concentration of reactants, concentration of salt, and upon temperature has been determined.

Tautomerism and Protonation of Guanosine

Guanosine has been demonstrated, by infrared and nuclear magnetic resonance spectroscopy, to have a keto-amino structure in neutral aqueous solution and to undergo protonation at N7 in acid solution.

Formation of a new 5′-guanylic acid helix in neutral solution

Abstract 5′-GMP forms in neutral solution a regular, ordered structure, presumably helical. The ordered structure is different from that previously reported at pH 5 and is not associated with gel formation. The transition is more cooperative than that of the pH 5 helix, but has a lower T m .

Non-enzymic oligonucleotide synthesis on a polycytidylate template.

Abstract A polycytidylate template facilitates both the self-condensation of guanosine-5′-phosphate and the condensation of guanosine-5′-phosphate with guanosine. It does not have any effect on the condensation of guanosine-5′-phosphate with other nucleosides. Guanosine-5′-phosphate condenses with itself less readily in its helical self-structure than in free solution.

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.

Poly 2-amino-6-N-methyladenylic acid: synthesis, characterization and interaction with polyuridylic acid.

Abstract A new polynucleotide containing adenylic acid residues modified by introduction of a 2-amino group and a 6- N -methyl group has been synthesized by enzymic polymerization of the nucleoside diphosphate. Properties of the polymer and its interaction products have been investigated by a variety of physical and spectroscopic methods. The polymer forms a non-regular stacked structure in neutral solution and a regular helical structure in acid. The former is more stable and the latter less stable thermally than the corresponding forms of poly A. In interesting contrast to the model proposed for acid poly A the acid helix of the new polymer cannot have an interchain amino-phosphate hydrogen bond. Both the nucleoside 2-amino-6- N -methyladenosine and poly 2NH 2 6Me A ‡ form 1:1 helical complexes with poly U. The effects of the 2-amino and 6- N -methyl groups on T m of the complexes with poly U are approximately additive and appear to be exerted independently of each other. The monomer-polymer helix provides a valuable model system for studying electrostatic interactions in polynucleotides since it has no interstrand electrostatic repulsion.

A displacement reaction between a polynucleotide helix and a random coil

The three-stranded helical complex of polyadenylic acid and polyinosinic acid, poly (A+ 2I), reacts with single-stranded polycytidylic acid to form a double-stranded helix, poly (I + C), with the release of single-stranded poly A according to the reaction, Poly (A + 2I) + 2 poly C → 2 poly (I + C) + poly A This strand displacement reaction was examined by ultraviolet and infrared spectroscopy of the reaction mixture and the reaction products were separated by sucrose gradient sedimentation and identified by ultraviolet spectroscopy. The reaction proceeds readily to completion at temperatures well below the T m of the less stable reactant helix, poly (A + 2I).

Tautomerism and Site of Protonation of 1-Methylcytosine: Proof by Nuclear Magnetic Resonance Spin-Spin Coupling

New nuclear magnetic resonance data lead to a completely unambiguous proof that the predominant tautomeric form of 1-methylcytosine is the amino form and that in acid solution the molecule protonates at 3-N. The close similarity of the nuclear magnetic resonance spectra of this compound to the spectra of the cytosine nucleosides indicates that these structures also exist in the nucleosides. These conclusions are reached on the basis of the spectra of analogs labeled with N15 and of the proton spin decoupling measurements made at low temperature.

Quantitative infrared spectra in D2O of some nucleosides, nucleotides, and polynucleotides: A new measure of polynucleotide interaction

Abstract Integrated infrared absorption intensities have been determined for some nucleosides, nucleotides, and polynucleotides in D2O solution. There are marked changes in the infrared spectra upon mixing the polynucleotides Poly A and Poly U. These changes are considered to result from hydrogen-bonding interaction between the two polynucleotides. The spectra indicate that the uracil residues in the nucleosides, nucleotides, polynucleotides, and in the mixture of polynucleotides are in the keto form and that the adenine units are probably in the amino form.