David R. Kearns

NMR Studies of Conformational States and Dynamics of DNA

The application of high resolution NMR techniques to the investigation of DNA double helices in solution is currently in a rapid state of change as a result of advances in three different fields. First, new methods (cloning, enzymatic degradation, sonication, and chemical synthesis) have been developed for producing large quantities of short DNA suitable for NMR studies. Second, there have been major advances in the field of NMR in terms of the introduction of new pulse techniques and improvements in instrumentation. Finally, as a result of recent X-ray diffraction studies on short DNA helices and the discovery of left-handed Z-DNA there is heightened interest in the study of DNA structures in solution and the effect of sequence on structure. In the present review, we discuss the way in which NMR techniques have been used to probe various aspects of the DNA properties, including base pairing structure, dynamics of breathing, effect of sequence on DNA structure, internal molecular motions, the effect of environment on the DNA, and the interaction of DNA with small ligands.

High-resolution nuclear magnetic resonance investigations of the structure of tRNA in solution.

Publisher Summary This chapter reviews that high-resolution NMR has been used to provide information on the secondary and tertiary structure of tRNA molecules in solution. It focuses on low field resonances from the ring nitrogen protons involved in base pairing, as these have provided most of the information to date. NMR complements and corroborates the recent X-ray diffraction studies on one particular tRNA, yeast tRNA Phe , and permits the crystal results on this one tRNA to be generalized to an entire class of tRNA. In this way, the NMR results have served as a bridge to justify the extension of the crystallographic results to tRNA Phe and other tRNAs in solution. The chapter also discusses that NMR can be used to treat tRNA structure and function problems that are currently unapproachable by X-ray diffraction analysis. NMR has been used to work out plausible structures for the denatured conformers of two tRNAs, and for the dimer of another. The alteration of tRNA conformation as a result of several different chemical modifications removal or replacement of bases in the anticodon loop has also been analyzed. NMR experiments have provided interesting insight into the interaction of tRNA molecules with intercalating drugs and dyes, which may find application in other biochemical and physical chemical studies of tRNA protein interactions.

SOLID-PHASE SYNTHESIS OF 5-HYDROXYMETHYLURACIL CONTAINING DNA

Abstract The synthesis of 3′-O-(diisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′-diemthoxytriyl)-5-( tert -butyldimethylsiloxymethyl)-2′-deoxyuridine ( 5 ) and its utilization for the preparation of 5-hydroxymethyluracil (hmU) containing oligodeoxyribonucleotides by means of automated synthesis are described.

Effect of magnesium and polyamines on the structure of yeast tRNAPhe.

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.

Interaction of water with oriented DNA in the A- and B-form conformations

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.

Hydrogen bonding of the 2' OH in RNA.

Abstract The high resolution PMR spectra of single and double stranded RNA and transfer RNA all exhibit resonances from exchangeable protons at about 6.8 ppm. This resonance at 6.8 ppm can be assigned to the 2′ OH since (i) resonances from all other exchangeable protons are otherwise accounted for, (ii) the resonance position is the same (±0.2 ppm) in all RNA indicating that it is from a proton in the backbone, (iii) the resonance is only observed from RNA at low temperature, and (iv) the resonance shifts upfield with the addition of dimethylsulfoxide to samples originally in water such that the extrapolation of the resonance position to neat dimethylsulfoxide is about 5.6 ppm which is approximately the resonance position of the 2′ OH proton of mononucleosides in dimethylsulfoxide. The 2′ OH is hydrogen bonded since the resonance position in RNA is shifted more than one ppm downfield from that of the mononucleoside in dimethylsulfoxide and the rate of exchange of the 2′ OH is considerably slower in RNA than in the monomer units. Examination of the PMR of mononucleosides in water dimethylsulfoxide mixtures, where the exchange is slow, shows that the resonance position of the 2′ OH hydrogen bonded to water is about 6.5 ppm. These PMR results indicate that the 2′ OH proton is hydrogen bonded to water. Examination of molecular models of RNA indicate a bound water molecule which can function as the acceptor of the 2′ OH hydrogen bond is properly situated to act as the donor in a hydrogen bond to the 3′ phosphate. The existence of this hydrogen bonding helps to explain some of the differences in conformation and thermal stability of RNA and DNA.

Static disorder and librational motions of the purine bases in films of oriented Li-DNA☆

Solid-state 2H nuclear magnetic resonance line shapes have been obtained from folded films of oriented Li-DNA molecules with the purine bases selectively labeled with deuterium at the 8-position. From line shape simulations, the static base tilts as well as the anisotropic motional amplitudes were determined as a function of hydration level and temperature. It was found that the average tilt angle of the bases is close to 0 degrees and at a hydration of ten water molecules per nucleotide the distribution width of tilt angles about this average cannot be larger than 9 degrees (standard deviation). A slightly increased distribution width is observed at low hydration levels. The motional amplitudes are hydration dependent, with the tilting motion ranging from 4 degrees for the driest, up to 15 degrees for the wettest sample, and slightly larger amplitudes are observed for the twisting motion. The amplitude of the twisting motion is unaffected by a temperature decrease down to -60 degrees C, in contrast to the tilting motion that is suppressed at low temperatures.

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.

Substituent effects on the binding of ethidium and its derivatives to natural DNA

The binding of eight ethidium derivatives to short (approximately 35 base-pair), random sequence DNA has been investigated using 1H-NMR. At 35 degrees C, all drugs cause upfield shifts of the DNA imino proton resonances characteristic of intercalative binding to DNA, but the line shapes vary significantly with the nature of the drug. The results confirm our previous proposal that removal of the amino group at position-3, but not at position-8, on the parent ethidium shortens the lifetime of the intercalative state (less than 1-2 ms at 35 degrees C). These results suggest that hydrogen-bonding interactions with the 3-NH2 group are involved in stabilization of the drug-DNA complex or that changes in charge distribution that accompany removal of the 3-NH2 group reduce the complex stability. The magnitude of the shift of the drug-DNA spectra indicates a slight preference for binding of the drugs adjacent to G X C base-pairs.

Two Dimensional NMR Investigation of the Structural Properties of DNAS

We describe the use of 2D NOE and truncated driven NOE (TNOE) spectroscopy to investigate structural features of three alternating homopolymer systems; poly(dA-dT), poly(dG-dC) (B- and Z-forms), and poly(dI-dC). Poly(dA-dT) and poly(dI-dC) have Watson-Crick base pairing, not Hoogsteen pairing as recently proposed for fibers. The B-form of poly(dG-dC), poly(dA-dT) and poly(dI-dC) exhibit qualitative structural features expected for B-family DNA. Specifically, all have anti-glycosidic torsional angles. This is contrasted with results from Z-form poly(dG-dC) which exhibits a syn-torsional angle as well as other differences in the 2D NOE spectrum. 2D NOE measurements on the decamer d(ATATCGATAT)2 in H2O were used to delineate the various spin lattice relaxation pathways (dipolar, exchange) of the imino protons in this molecule.