Determination of Nucleic Acid Structures

The structure of the major photoproduct of irradiation of the dinucleotide d(TpA) and TA containing oligonucleotides has been re-characterised. 2 -Deoxyoxoanosine (218) has been isolated from calf thymus DNA after treatment with the mutagens nitrous acid or nitric oxide.  

The structural basis of phosphoramide mustard crosslinking to guanosines in an oligonucleotide has been studied both in vitro and by computer simulation. The effects of 5-fluorouridine and 5-fluoro-2 -deoxyuridine substitution into synthetic RNA and DNA have been studied. Destabilisation of duplex oligo-deoxynucleotides but stabilisation of oligoribonucleotides was found upon the analogue substitution for uridine in the parent duplexes.  

The role of dihydrouridine in RNA structures has been examined by NMR spectroscopy. It was found that the dihydro derivative introduced greater conformational flexibility to the RNA by promoting the C2 -endo sugar con- 

Jones and co-workers have used N7-labelled 2 -deoxyguanosine and 2 -deoxyadenosine incorporated into a synthetic oligonucleotide triplex stucture to study the hydrogen bonding at these sites. Hoogsteen hydrogen bonding to the N7 purine atoms was observed.  

Jovin and Jareserijman have used fluorescence resonance energy transfer to determine the helical handedness, twist and rise of different DNA conforma-tions. The technique relies on the attachment of two fluorescent dyes, a donor and an acceptor, to the DNA. 

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Describes the application of NMR to determination of nucleic acid structure. 

I here are numerous problems associated with the determination of nucleic acid structures in solution using NMR methods. These include spin diffusion, anisotropic rotation, conformational averaging, low density of experimental constraints and lack of long-range information. 

Yet, distance constraints or restraints are the most commonly used for the determination of nucleic acid structures from NMR experiments. However, this topic is much too large to be reviewed here and we will refer the reader to reviews (see also Macromolecular Structures Determined using NMR Data and NMR Refinement). 

This chapter describes the chemistry of nucleotides and the m or classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules. 

The lack of long-range restraints has hindered the precise and accurate structure determination of nucleic acids for a long time. The isotopic enrichment mostly improved the accuracy of local geometry without a pronounced influence on the overall shape. However,... 

The dependence of the residual dipolar coupling on the angle that the vector forms with a reference axis explains why the use of dipolar couplings makes possible the determination of the relative orientation of different domains in a multidomain protein and facilitates nucleic acid structure determination. Dipolar couplings can constitute up to 50% of the total structural data available for nucleic acids, while this number drops to 10-15% in proteins. Thus, the impact of the use of dipolar couplings on the structure determination of nucleic acids is generally more substantial than in the case of proteins. Furthermore, the presence or absence of tertiary structure in a protein or nucleic acid does not have a major influence on the number of dipolar couplings that can be measured, in contrast to the case of the NOE. 

For molecular sizes that are amenable by NMR techniques, nucleic acids usually lack a tertiary fold. This fact, together with the characteristic low proton density, complicates NMR structural analysis of nucleic acids. As a result, local geometries and overall shapes of nucleic acids, whose structures have been determined by NMR, usually are poorly defined. Dipolar couplings provide the necessary long-range information to improve the quality of nucleic acid structures substantially [72]. Some examples can be found already in the literature where the successful application of dipolar couplings into structure calculation and structure refinement of DNA and RNA are reported [73-77]. 

In the past most of the studies on structure determination of nucleic acids were performed by proton NMR [513, 767, 768]. However, recent 13C NMR investigations have demonstrated that additional information can be obtained from the 13C spectra [769]. 

The diffraction pattern to be expected for a helical structure was worked out in a theoretical study by William Cochran, Francis H. C. Crick, and Vladimir Vand using the a helix as a model. This work provided the basis for the interpretation of the diffraction patterns of proteins, and also led, unexpectedly, to an understanding of nucleic acid structure. This culminated in the determination of the three-dimensional structure of DNA by James D. Watson and Frances H. C. Crick from an X-ray diffraction photograph taken by Rosalind Franklin. 

HETCOR113 or hetero-TOCSY 114,115 see also a review by Pardi87 and references therein. Unfortunately, these methods are not routinely used for structure determination of nucleic acids, except for relatively short oligonucleotides, because of the relatively small chemical shift dispersion of 31P and its fast relaxation via the chemical shift anisotropy mechanism. 

R595 H. Zhou, A. Vermeulen, F. M. Jucker and A. Pardi, Incorporating Residual Dipolar Couplings into the NMR Solution Structure Determination of Nucleic Acids , Biopolymers, 1999-2000, 52, 168... 

The structure of an oligonucleotide designed to assemble a 4-way junction has been investigated by Preliminary results in the structure determination of nucleic acid analogues containing... 

Structure determination of nucleic acids faces several problems, which stem mostly from low proton density, lack of long-range restraints due to an elongated shape of majority structures, and a small number of NOE restraints between the distant elements of secondary structure. These difficulties can be circumvented by applying recently introduced technology for obtaining additional structural... 

DNA and RNA are complex biomacromolecules whose structures and interactions are intensively studied mainly in relation to their biological roles. The main interest of scientists is not oriented to the determination of nucleic acids in biological materials but to analysis of their nucleotide sequence, secondary and tertiary structures, specific interactions with proteins, etc. A number of diverse techniques are necessary such as absorption spectroscopy, chromatography, gel electrophoresis, and radioactive and nonradioactive labeling, which can be found in almost every nucleic acid laboratory, while other less universal techniques are applied in relation to the specific aims of the given laboratory. Electrochemical methods briefly reviewed in this article belong to the latter category. They can be extremely useful for a number of purposes, such as determination of traces... 

Westhof, E. Auffinger, P. in Molecular Modeling and Structure Determination of Nucleic Acids , Leontis, N. Santa Lucia Jr., J., Eds. American Chemical Society Washington, D.C., 1997. 

This chapter describes practical aspects of the application of UV absorbance temperature profiles to determine the thermodynamics of nucleic acid structural transitions. Protocols and practical advice are presented for issues not normally addressed in the primary literature but that are crucial for the determination of reliable thermodynamics, such as sequence design, sample preparation, choice of buffer, protocols for determining strand concentrations and mixing strands, design of microvolume cuvettes and cell holder, instrumental requirements, data analysis methods, and sources of error. References to the primaiy literature and reviews are also provided where appropriate. Sections of this chapter have been adapted from previous reviews and are reprinted with permission from the Annual Review of Biochemistry, Volume 62 1993, by Annual Reviews wwwAnnualReviews.org (6) and with permission from Biopolymers 1997, by John Wiley Sons, Inc. (4). 

Over the past few years there has been a significant increase in the number of nucleic acid structures reported. As advances in respective technologies arise, particularly with NMR spectroscopy, so the complexity of systems studied increases. However, more recently there are many new techniques that are being explored to aid structure determination, at least in global terms rather than at the atomic level, of larger and more complex nucleic acid systems. 

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