Structural Studies Design and Protocols

Structural Studies Design / Protocol

Nucleic acid structural studies cover a variety of topics, and typically require both modified and unmodified oligonucleotides for use as research tools in the particular study under question. The specific type, or types, of modifications required is highly dependent on the hypothesis being tested in the study.

Modifications such as 7-deaza-dG, 7-deaza-8-aza-dG, etc.) are used to investigate the effect on the activity of an oligonucleotide by an enzyme (e.g., polymerases, reverse transcriptases, restriction enzymes, etc.) when key structural elements are changed. These modifications typically have properties (hydrogen bonding, base-stacking, etc) that are significantly different from those of the standard nucleotide bases, which can be used to probe structure-activity relationships between the oligo and an enzyme.

Halogenated nucleotides (brominated, iodinated) are useful both in crystallography studies of oligonucleotide structure and in cross-linking studies to probe protein-nucleic acid complex structure.

Etheno-dA and Pyrrolo-dC are two modifications with fluorescent properties that make them useful as probes into DNA structure and dynamics. Etheno-dA is often used to observe transitions between DNA structural types. Pyrrolo-dC base pairs with G like the standard dC nucleotide, and its small size does not disturb the double-helix. It has markedly different fluorescent levels in single-stranded vs. double-stranded DNA, so it is particularly useful for studying regions of local duplex melting.

References

(1) Srivastava, S.C., Raza, S.K., Misra, R. 1,N6-etheno deoxy and ribo adenoGine and 3,N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules. Nucleic Acids Res. (1994), 22: 1296-1304.
(2) Dosanjh, M.K., Roy, R., Mitra, S., Singer, B. 1, N6-ethenoadenine is preferred over 3-methyladenine as substrate by a cloned human N-methylpurine-DNA glycosylase (3-methyladenine-DNA glycosylase). Biochemistry (1994), 33: 61624-1628.
(3) Chung, F-L., Chen, H-J.C., Nath, R.G. Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis (1996), 17: 2105-2111.
(4) Kandimalla, E.R., Yu, D., Zhao, Q., Agrawal, S. Effect of chemical modifications of cytosine and guanine in a CpG-motif of oligonucleotides: structure-immunostimulatory activity relationships. Bioorg. Med. Chem. (2001), 9: 807-813.