Nucleic acids structural analysis

Turning to nucleic acids, the potential of UV-visible spectroscopy for nucleic acid structural analysis has always been considered high in principle. The reason for this is the sheer number of base chromophores available in nucleic acids and their potential for electronic interactions. Given the high degree of structural uniformity in nucleic acids, the total absorbance A(A.) of a given sample of nucleic acids may be given by... 

M. S. Babcock, E. P. D. Pendault, and W. K. Olson,/ Mol. Biol., 237,125 (1994). Nucleic Acid Structure Analysis. Mathematics for Local Cartesian and Helical Structure Parameters That Are Truly Comparable Between Structures. 

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]. 

The intercalation of polycyclic aromatic compounds into duplex DNA structures was used to develop nucleic acid-based electrochemical sensors.66 For example, the bis-ferrocene-tethered naphthalene diimide (16) was used as a redox-active intercalator to probe DNA hybridization.67 The thiolated probe was assembled on a Au electrode, and the formation of the duplex DNA with the complementary analyte nucleic acid was probed by the intercalation of (16) into the double-stranded nucleic acid structure and by following the voltammetric response of the ferrocene units (Fig. 12.17a). The method enabled the analysis of the target DNA with a sensitivity that corresponded to ca. 1 x 10-20mol. 

The versatility of agarose gels is obvious when one reviews their many applications in nucleic acid analysis. The rapid advances in our understanding of nucleic acid structure and function in recent years are due primarily to the development of agarose gel electrophoresis as an analytical tool. Two of the many applications of agarose gel electrophoresis will be described here. 

Lilley, D. M. J. (2008). Analysis of branched nucleic acid structure using comparative gel electrophoresis. Quart. Rev. Biophys. 41(1), 1—39. 

Human apoA-I is a major constituent of HDL, with an Mr of approximately 28,300, calculated from the known primary structure (Bl, B43). ApoA-I is initially synthesized as a 267-amino-acid precursor protein, pre-pro-apoA-I (G25, G26), containing an 18-amino-acid prepeptide and a 6-amino-acid propeptide [determined by nucleic acid sequence analysis of cloned apoA-I (L6), and by isolating the primary translation product of human intestinal apoA-I mRNA (G25)]. 

The structures of the rRNAs were generated and sequences compared using the University of Wisconsin Genetics Computer Group Nucleic Acid sequence analysis package29 and the University of Georgia Biological Structure and Sequence Computation Facility. 

Most of our structural information comes from x-ray crystallographic analysis of protein crystals and from the use of nuclear magnetic resonance spectroscopy in solution. Each of these techniques has advantages and limitations which makes them suitable for a complementary range of problems. The first protein structure determined at a sufficient resolution to trace the path of the polypeptide chain was that of myoglobin in 1960. Since that time many thousands of structures corresponding to hundreds of different proteins have been determined. The coordinates of the atoms in many protein and nucleic acid structures are available from the Protein Data Bank, which may be accessed via the Internet or World Wide Web (http //www.pdb.bnl.gov). 

Arnott S (1981) The secondary structures of polynucleotide chains as revealed by X-ray diffraction analysis of fibers. In Neidle S (ed) Tbpics in nucleic acid structure. MacMillan, London, pp 65-82... 

Sequence similarity database searching and protein sequence analysis constitute one of the most important computational approaches to understanding protein structure and function. Although most computational methods used for nucleic acid sequence analysis are also applicable to protein sequence studies, how to capture the enriched features of amino acid alphabets (Chapter 6) poses a special challenge for protein analysis. 

X-ray crystallographic analyses usually provide structural information that correctly represents the actual structures of metal ion-nucleic acid complexes in the crystal this usually represents an environment that is drastically different from normal solution conditions. Obviously, such analyses are limited to complexes that are both stable and amenable to crystallization. In comparison, solution NMR spectroscopy data are much more generally applicable to the analysis of metal ion-nucleic acid structure, " " but can be more easily misinterpreted owing to factors such as the distribution of metal ions among several binding sites and the intrinsically weaker nature of some of the interactions that are studied. Also, nucleic acid crystallization requires the formation of ordered and uniform intermolecular interactions between nucleic acids. These intermolecular interactions may either compete or interfere with the formation of certain metal-nucleic complexes and promote the formation of unnatural metal binding centers. ... 

Lu X-J, Olson WK. 3DNA A software package for the analysis, 38. rebuilding, and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 2003 31 5108-5121. 

As in previous years, there is a growing number of nucleic acid structures reported. Advances in X-ray and NMR methodologies means that more complex structures are now being studied. However, in addition to these more traditional methods of structure analysis there are also other techniques emerging, and these are discussed at the end of this section. 

Holmes, K. C., and Blow, D. M. The Use of X-ray Diffraction in the Study of Protein and Nucleic Acid Structure. Revised reprint from Methods of Biochemical Analysis. (Ed., Glick, D.) 13, 113-239 (1966). Interscience (John Wiley) New York, London, Sydney (1966). Reprinted Krieger Melbourne, EL (1979). 

Notwithstanding this complexity, the need for three-dimensional, structural information at the atomic level of resolution is central and indispensable to biomembrane science. X-ray, and to a lesser extent neutron-diffraction, as the most important sources for such information have, therefore, been widely used in this field (for reviews, see Refs. 1-4). The success of this approach, however, has generally been less spectacular than for instance in the cases of protein or nucleic acid structure. The reasons for this lie in the very nature of biological membranes with few, notable exceptions (such as the purple membrane of halobacterium halobium, which can be viewed essentially as a two-dimensional crystal of bacteriorhodopsin with only little lipid. Refs. 5, 6,25) biological membranes are characterized by highly complex and variable molecular compositions, and by the structural dynamics, fluidity , which is in many cases essential for enzymatic, or other, functions of membranes. As a reflection of this most natural membranes do not crystallize, and a full, three-dimensional atomic structure analysis seems out of reach. 

Computational proteomics refers to the large-scale generation and analysis of 3D protein structural information. Accurate prediction of protein contact maps is the beginning and essential step for computational proteomics. The major resource for computational proteomics is the currently available information on protein and nucleic acid structures. The 3D-GENOM1CS (www.sbg.bio.ic.au.uk/3dgenomics/) and PDB (http // www.rcsb.org/pdb/), and other databases provide a broad range of structural and functional annotations for proteins from sequenced genomes and protein 3D structures, which make a solid foundation for computational proteomics. 

Transfer RNA, tRNA, soluble RNA, sRNA. Low mol wt 23,000-27,000 approx 75-85 nucleotides. Each tRNA is specific for and binds with a particular amino acid more than one may exist for each amino acid. Performs three functions during protein synthesis binds with its specific amino acid recognizes the corresponding codon on mRNA and places the amino acid in the correct position for attach -ment to the polypeptide chain being formed binds the poly -peptide to the ribosome. First determination of total structure of a transfer RNA (yeast alanine tRNA) Holley et aL. Science 147, 1462 (1965). Reviews of structure and function Miura, Specificity in the Structure of Transfer RNA in fVogr, Nucleic Acid Res. Mol. BioL 6, 39-82 (1967) Cramer, Three-Dimensional Structure of tRNA , ibid. 11, 391-421 (1971) Nucleic Acid Sequence Analysis, S. Mandeles (Columbia University Press, New York, 1972) pp 256-280 Nishi-mura, "Transfer RNA Structure and Biosynthesis in MTP Int. Rev. Sci Biochem.. Ser. One vol. 6, K. Burton, Ed. (University Park Press, Baltimore, 1974) pp 289-322 A. Rich, V. L. Raj Bhandary, Ann. Rev. Biochem. 45, 805-860 (1976) P. F. Agris, The Modified Nucleosides of Transfer RNA, IT (A. R. Liss, New York, 1983) 220 pp. 

A third area is the statistical analysis of experimental structure sets. Even though the number of experimentally known three-dimensional nucleic acid structures is still rather small as compared to proteins the statistical analysis and search for structural motifs becomes more an more important also for nucleic acids and nucleic acid/protein complexes. ... 

This table takes only structures into account for which atomic coordinates have been deposited at the Protein Data Bank. Given polyads occur in several chain this indicated before the polyad. If polyads include bases from different chains the chain identifier is indicated in parenthesis. The numbering of bases involved in polyads is not given for regular triplex/tetr lex structures. The topology corresponds to the classification presented in Table 1. H-bonds between bases are indicated by dots. No difference is made between Watson-Crick and non-canonical base-base interactions. The base sequence follows the H-bond interaction pattern. For example, in the non-cyclic triad C.G.A C is bound to G and G to A. In more complex cases parentheses have been used. The H-bond analysis of nucleic acid structures has been performed with HBexplore. 

Common structural elements can be identified using mFold, a program that predicts secondary structure of ssDNA or RNA." For this analysis, it is necessary to include the primer regions since they most likely contribute to the overall structure of the nucleic acid. This analysis is useful in identifying common structural elements in aptamers even if they are generated by differing sequences. 

In another approach, He et al. (He et al., 2013) proposed a 2-site per nucleotide (NARES-2P, nucleic acid united residue 2-point model) CG model where chain connectivity, excluded volume and base dipole interactions are sufficient to form helical DNA and RNA structures. This model was parametrized using a bottom-up strategy by employing a set of statistical potentials, derived from DNA and RNA structures from the Protein Data Bank, and the Boltzmann inversion method to reproduce the structural features. The base-base interactions were parametrized by fitting the potential of mean force to detailed all-atoms MD simulations using also the Boltzmann inversion approach. The respective potentials do not explicitly define the nucleic-acid structure, dynamics and thermod3mamics, but are derived as potentials of mean force. By detailed analysis of the different contribution to the Hamiltonian, the authors determined that the multipole-multipole interactions are the principal factor responsible for the formation of regular structures, such as the double helical structures. 

DNA structure (Wilkins and Franklin), and the structure determination of vitamin B12 (Hodgkin). All these structure determinations are of varying complexity, but the most remarkable achievements of twentieth century x-ray analysis lie in the fields of protein, enzyme and nucleic acid structure (Section 14.3). 

Structure. The mononucleotides of RNA consist of ribose phosphorylated at C3, and linked by an N-gly-cosidic bond to one of four bases adenine, guanine, cytosine or uracil. Many other bases (chiefly methylated bases) also occur, but are less common (see Rare nucleic acid components). The mononucleotide units form a linear chain via 3, 5 phosphodiesler bonds (see Nucleic acids). Sequence analysis of RNA has become a standard technique. In many cases the amino acid sequences of proteins are predicted from the sequence of the corresponding mRNA (or DNA) because it is much easier to clone the nucleic acid than to isolate the protein. 

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). 

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