Diffusion-ordered nuclear magnetic resonance spectroscopy for analysis of DNA secondary structural elements

Abstract

Structure determination of secondary DNA structural elements, such as G-quadruplexes, gains an increasing importance as fundamental physiological roles are being associated with the formation of such structures in vivo. A truncated native DNA sequence generally requires further optimization to obtain a candidate with desired NMR properties for structural analysis in solution. The optimum sequence is expected to form one dominant, stable molecular entity in solution with well-resolved NMR peaks. However, DNA sequences are prone to form structures composed of one, two, three or four strands depending on sequence and solution conditions. The thorough characterization of the molecularity (stoichiometry and molecular weight) and appropriate solution conditions for sequences with different modifications traditionally applies analytical techniques that generally do not represent the solution conditions for NMR structure determination. Here we present the application of diffusion ordered NMR spectroscopy as a useful analytical tool for the optimization and analysis of DNA secondary structural elements, specifically, the DNA G-quadruplex structures, including those formed in the human telomeric sequence and in the promoter regions of bcl-2 and c-myc genes. KeywordsDNA; NMR; DOSY; quadruplex; bcl-2; human telomere; c-myc Structural analysis of unusual DNA motifs with particular emphasis on G-quadruplexes and imotifs has a central role in the identification and characterization of molecular targets related to these structures [1][2][3][4][5][6][7]. Such structures are implicated in several physiologically important regulatory processes, with special emphasis on carcinogenesis [4][5][6][7][8][9][10][11][12]. Most of the structural studies on these elements have been performed by NMR spectroscopy. In most cases the related nucleotide sequence bearing the actual physiological function in vivo needs to be truncated/ elongated and/or mutated in order to obtain a successful candidate for NMR structure determination [3][4][5][6][7]13]. This NMR candidate should be a well-defined molecular entity in physiologically relevant solution conditions (ionic strength, pH, temperature) representing the same conformation as the original sequence. Appropriate modification of the sequence by mutation/truncation/elongation will force the molecule to form a single stable structure without altering crucial connectivities (conformation) of the structure.* To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel St., P.O. Box 210207, room 101/406, Tucson, Arizona 85721, USA; Tel: +1 520 626 6749/626 5969, Fax: +1 520 626 2466, E-mail: ambrus@pharmacy.arizona.edu, yangd@pharmacy.arizona.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of ...

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

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Rapid Stoichiometric Analysis of G‐Quadruplexes in Solution

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“…The human telomeric sequence dA C H T U N G T R E N N U N G [AG 3 A C H T U N G T R E N N U N G (TTAG 3 ) 7 TTA] comprises eight guanine segments. The single peak in the average diffusion profiles for the telomeric sequence, for both the disordered state, and in 120 mm potassium, suggests a single species (Figure 1 B and C), respectively.…”

The Use of a Peptidic Scaffold for the Formation of Stable Guanine Tetrads: Control of a H‐bonded Pattern in Water

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