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. 1988 Sep;85(17):6242–6246. doi: 10.1073/pnas.85.17.6242

Influence of loop residues on the relative stabilities of DNA hairpin structures.

M M Senior 1, R A Jones 1, K J Breslauer 1
PMCID: PMC281945  PMID: 3413094

Abstract

We have determined the relative stabilities and melting behaviors of DNA hairpin structures as a function of the nonbonded residues in the loop. The specific family of hairpin structures we investigated in this work is formed by the 16-mer sequence d[CGAACG(X)4CGTTCG], where X is deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine. As shown below, this 16-mer can fold back on itself to form a family of DNA hairpin structures that possess a common hexameric stem duplex and a nonbonded loop of 4 nucleotides. For the hairpin structures investigated in this work, we varied the loop composition from all purine residues to all pyrimidine residues. (Formula: see text). We thermodynamically characterized the relative stabilities and melting profiles of these hairpin structures by a combination of spectroscopic and calorimetric techniques. To establish a thermodynamic "baseline," we also conducted parallel studies on the isolated hexameric duplex d[CGAACG).(CG-TTCG)], which corresponds to the common stem duplex present in each hairpin structure. Our spectroscopic and calorimetric data reveal the following: (i) The hairpin structure with four dT residues in the loop exhibits the highest melting temperature, while the corresponding hairpin structure with four dA residues in the loop exhibits the lowest melting temperature. (ii) The free energy data at 25 degrees C reveal the following order of DNA hairpin stability for the four structures studied here: T loop greater than C loop greater than G loop greater than A loop. In other words, the pyrimidine-looped hairpins of four residues are more stable than the purine-looped hairpins. (iii) The loop-dependent order of hairpin stability is paralleled by a similar trend in the calorimetrically determined transition enthalpies for hairpin disruption. Thus, the enhanced stability of the pyrimidine-looped hairpin structures relative to purine-looped hairpin structures is enthalpic in origin. To develop insight into the molecular basis for the thermodynamic differences, proton NMR spectroscopy was used to probe for structural disparities between the most stable hairpin structure (T loop) and the least stable hairpin structure (A loop). Two-dimensional nuclear Overhauser enhancement spectroscopy revealed connectivities between the residues in the stem duplexes of both hairpin structures that are consistent with B-form DNA. In addition, the nonbonded residues in both the T and A loops exhibited the same connectivity patterns. However, on the 5' side of the stem-loop junction, the T-loop residue exhibited a connectivity with the adjacent base pair of the stem duplex that is not observed for the corresponding A-loop residue. This difference in connectivities at the stem-loop junction may provide a structural basis for our observation that the T-looped hairpin structure is more stable than the corresponding A-looped hairpin structure.

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Selected References

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  1. Albergo D. D., Marky L. A., Breslauer K. J., Turner D. H. Thermodynamics of (dG--dC)3 double-helix formation in water and deuterium oxide. Biochemistry. 1981 Mar 17;20(6):1409–1413. doi: 10.1021/bi00509a001. [DOI] [PubMed] [Google Scholar]
  2. Borer P. N., Dengler B., Tinoco I., Jr, Uhlenbeck O. C. Stability of ribonucleic acid double-stranded helices. J Mol Biol. 1974 Jul 15;86(4):843–853. doi: 10.1016/0022-2836(74)90357-x. [DOI] [PubMed] [Google Scholar]
  3. Breslauer K. J., Frank R., Blöcker H., Marky L. A. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. doi: 10.1073/pnas.83.11.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breslauer K. J., Sturtevant J. M., Tinoco I., Jr Calorimetric and spectroscopic investigation of the helix-to-coil transition of a ribo-oligonucleotide: rA7U7. J Mol Biol. 1975 Dec 25;99(4):549–565. doi: 10.1016/s0022-2836(75)80171-9. [DOI] [PubMed] [Google Scholar]
  5. Chu Y. G., Tinoco I., Jr Temperature-jump kinetics of the dC-G-T-G-A-A-T-T-C-G-C-G double helix containing a G . T base pair and the dC-G-C-A-G-A-A-T-T-C-G-C-G double helix containing an extra adenine. Biopolymers. 1983 Apr;22(4):1235–1246. doi: 10.1002/bip.360220415. [DOI] [PubMed] [Google Scholar]
  6. Frank-Kamenetskii M. D., Vologodskii A. V. Thermodynamics of the B-Z transition in superhelical DNA. Nature. 1984 Feb 2;307(5950):481–482. doi: 10.1038/307481a0. [DOI] [PubMed] [Google Scholar]
  7. Gaffney B. L., Marky L. A., Jones R. A. Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine. Biochemistry. 1984 Nov 20;23(24):5686–5691. doi: 10.1021/bi00319a004. [DOI] [PubMed] [Google Scholar]
  8. Gralla J., Crothers D. M. Free energy of imperfect nucleic acid helices. II. Small hairpin loops. J Mol Biol. 1973 Feb 5;73(4):497–511. doi: 10.1016/0022-2836(73)90096-x. [DOI] [PubMed] [Google Scholar]
  9. Gralla J., DeLisi C. mRNA is expected to form stable secondary structures. Nature. 1974 Mar 22;248(446):330–332. doi: 10.1038/248330a0. [DOI] [PubMed] [Google Scholar]
  10. Grosschedl R., Hobom G. DNA sequences and structural homologies of the replication origins of lambdoid bacteriophages. Nature. 1979 Feb 22;277(5698):621–627. doi: 10.1038/277621a0. [DOI] [PubMed] [Google Scholar]
  11. Haasnoot C. A., de Bruin S. H., Berendsen R. G., Janssen H. G., Binnendijk T. J., Hilbers C. W., van der Marel G. A., van Boom J. H. Structure, kinetics and thermodynamics of DNA hairpin fragments in solution. J Biomol Struct Dyn. 1983 Oct;1(1):115–129. doi: 10.1080/07391102.1983.10507429. [DOI] [PubMed] [Google Scholar]
  12. Hare D. R., Reid B. R. Three-dimensional structure of a DNA hairpin in solution: two-dimensional NMR studies and distance geometry calculations on d(CGCGTTTTCGCG). Biochemistry. 1986 Sep 9;25(18):5341–5350. doi: 10.1021/bi00366a053. [DOI] [PubMed] [Google Scholar]
  13. Hilbers C. W., Haasnoot C. A., de Bruin S. H., Joordens J. J., van der Marel G. A., van Boom J. H. Hairpin formation in synthetic oligonucleotides. Biochimie. 1985 Jul-Aug;67(7-8):685–695. doi: 10.1016/s0300-9084(85)80156-5. [DOI] [PubMed] [Google Scholar]
  14. Ikuta S., Chattopadhyaya R., Ito H., Dickerson R. E., Kearns D. R. NMR study of a synthetic DNA hairpin. Biochemistry. 1986 Aug 26;25(17):4840–4849. doi: 10.1021/bi00365a018. [DOI] [PubMed] [Google Scholar]
  15. Leng M., Felsenfeld G. A study of polyadenylic acid at neutral pH. J Mol Biol. 1966 Feb;15(2):455–466. doi: 10.1016/s0022-2836(66)80121-3. [DOI] [PubMed] [Google Scholar]
  16. Lilley D. M. Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property. Nucleic Acids Res. 1981 Mar 25;9(6):1271–1289. doi: 10.1093/nar/9.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lilley D. M. The inverted repeat as a recognizable structural feature in supercoiled DNA molecules. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6468–6472. doi: 10.1073/pnas.77.11.6468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marky L. A., Blumenfeld K. S., Kozlowski S., Breslauer K. J. Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): evidence for hairpin formation. Biopolymers. 1983 Apr;22(4):1247–1257. doi: 10.1002/bip.360220416. [DOI] [PubMed] [Google Scholar]
  19. Marky L. A., Breslauer K. J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers. 1987 Sep;26(9):1601–1620. doi: 10.1002/bip.360260911. [DOI] [PubMed] [Google Scholar]
  20. Martin F. H., Uhlenbeck O. C., Doty P. Self-complementary oligoribonucleotides: adenylic acid-uridylic acid block copolymers. J Mol Biol. 1971 Apr 28;57(2):201–215. doi: 10.1016/0022-2836(71)90341-x. [DOI] [PubMed] [Google Scholar]
  21. Orbons L. P., van der Marel G. A., van Boom J. H., Altona C. Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study. Nucleic Acids Res. 1986 May 27;14(10):4187–4196. doi: 10.1093/nar/14.10.4187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Panayotatos N., Wells R. D. Cruciform structures in supercoiled DNA. Nature. 1981 Feb 5;289(5797):466–470. doi: 10.1038/289466a0. [DOI] [PubMed] [Google Scholar]
  23. Patel D. J., Shapiro L., Hare D. Nuclear magnetic resonance and distance geometry studies of DNA structures in solution. Annu Rev Biophys Biophys Chem. 1987;16:423–454. doi: 10.1146/annurev.bb.16.060187.002231. [DOI] [PubMed] [Google Scholar]
  24. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  25. Scheffler I. E., Elson E. L., Baldwin R. L. Helix formation by d(TA) oligomers. II. Analysis of the helix-coli transitions of linear and circular oligomers. J Mol Biol. 1970 Feb 28;48(1):145–171. doi: 10.1016/0022-2836(70)90225-1. [DOI] [PubMed] [Google Scholar]
  26. Scheffler I. E., Elson E. L., Baldwin R. L. Helix formation by dAT oligomers. I. Hairpin and straight-chain helices. J Mol Biol. 1968 Sep 28;36(3):291–304. doi: 10.1016/0022-2836(68)90156-3. [DOI] [PubMed] [Google Scholar]
  27. Sinden R. R., Pettijohn D. E. Cruciform transitions in DNA. J Biol Chem. 1984 May 25;259(10):6593–6600. [PubMed] [Google Scholar]
  28. Summers M. F., Byrd R. A., Gallo K. A., Samson C. J., Zon G., Egan W. Nuclear magnetic resonance and circular dichroism studies of a duplex--single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG). Nucleic Acids Res. 1985 Sep 11;13(17):6375–6386. doi: 10.1093/nar/13.17.6375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tan Z. K., Ikuta S., Huang T., Dugaiczyk A., Itakura K. Solid-phase synthesis of polynucleotides. VIII: A simplified synthesis of oligodeoxyribonucleotides. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):383–391. doi: 10.1101/sqb.1983.047.01.045. [DOI] [PubMed] [Google Scholar]
  30. Tsong T. Y., Hearn R. P., Wrathall D. P., Sturtevant J. M. A calorimetric study of thermally induced conformational transitions of ribonuclease A and certain of its derivatives. Biochemistry. 1970 Jun 23;9(13):2666–2677. doi: 10.1021/bi00815a015. [DOI] [PubMed] [Google Scholar]
  31. Uhlenbeck O. C., Borer P. N., Dengler B., Tinoco I., Jr Stability of RNA hairpin loops: A 6 -C m -U 6 . J Mol Biol. 1973 Feb 5;73(4):483–496. doi: 10.1016/0022-2836(73)90095-8. [DOI] [PubMed] [Google Scholar]
  32. Warshaw M. M., Cantor C. R. Oligonucleotide interactions. IV. Conformational differences between deoxy- and ribodinucleoside phosphates. Biopolymers. 1970;9(9):1079–1103. doi: 10.1002/bip.1970.360090910. [DOI] [PubMed] [Google Scholar]
  33. Wells R. D., Goodman T. C., Hillen W., Horn G. T., Klein R. D., Larson J. E., Müller U. R., Neuendorf S. K., Panayotatos N., Stirdivant S. M. DNA structure and gene regulation. Prog Nucleic Acid Res Mol Biol. 1980;24:167–267. doi: 10.1016/s0079-6603(08)60674-1. [DOI] [PubMed] [Google Scholar]
  34. Wemmer D. E., Chou S. H., Hare D. R., Reid B. R. Duplex-hairpin transitions in DNA: NMR studies on CGCGTATACGCG. Nucleic Acids Res. 1985 May 24;13(10):3755–3772. doi: 10.1093/nar/13.10.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wickstrom E., Tinoco I., Jr The stability of RNA hairpin loops containing A-U-G: An-U-G-Um. Biopolymers. 1974 Nov;13(11):2367–2383. doi: 10.1002/bip.1974.360131116. [DOI] [PubMed] [Google Scholar]
  36. Xodo L. E., Manzini G., Quadrifoglio F., van der Marel G. A., van Boom J. H. Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution. Nucleic Acids Res. 1986 Jul 11;14(13):5389–5398. doi: 10.1093/nar/14.13.5389. [DOI] [PMC free article] [PubMed] [Google Scholar]

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