Conformational study of biotinylated DNA oligonucleotides utilizing two-dimensional nuclear magnetic resonance and molecular dynamics.

The solution structure of DNA oligonucleotides containing a biotin group covalently attached through a linker arm to the 5' end has been studied by two-dimensional NMR and molecular modeling, in an attempt to determine whether the biotin end group is accessible to avidin binding. Such experiments are useful in suggesting that purification of synthetic DNA oligonucleotides by avidin-biotin affinity chromatography is carried out with a minimum amount of intramolecular association of biotin with potential binding sites within the DNA. Two DNA oligonucleotides that form hairpin structures have been proposed as possible worst case scenarios where biotin would not be available for avidin binding. These structures are d(XGCGCGTTTTCGCGC) and d(XGCGCGTTTTCGCGCAAAAA), where X represents the biotin and linker structures. No NOEs were detected between the biotin/linker portion of the molecule and the DNA hairpin. In addition, the magnitude of the NOEs between neighboring groups in the biotin/linker was approximately an order of magnitude smaller than that of the neighbors at a comparable distance within the DNA hairpin, suggesting that the biotin/linker undergoes considerable additional motion compared to the DNA hairpin. Molecular dynamics calculations also show that the biotin/linker undergoes a considerable range of motion. Thus, all data indicate that the biotin is not immediately associated with the hairpin and should be available for binding to avidin under chromatographically relevant solvent conditions.

Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5.

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.

Characterization of anti-Z-RNA polyclonal antibodies: epitope properties and recognition of Z-DNA.

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)