Engineering Terminal Disulfide Bonds Into DNA

Gary D. Glick1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.7
DOI:  10.1002/0471142700.nc0507s13
Online Posting Date:  August, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This protocol presents a simple and general means of modifying nucleic acids with disulfide cross‐links. These cross‐links serve as powerful tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross‐links have been used to study proteins. The chemistry described has been used to synthesize disulfide‐cross‐linked hairpins and duplexes, higher‐order structures such as triplexes, non‐ground‐state conformations, and tRNAs. since the cross‐links form quantitatively by mild air oxidation and do not purturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids.

Keywords: nucleic acid; disulfide cross‐link; structure; dynamics; stability

PDF or HTML at Wiley Online Library

Table of Contents

  • Design of an Ideal Cross‐Link
  • Disulfide Cross‐Linked DNA Duplexes
  • Disulfide Cross‐Linked DNA Triplexes
  • Applications
  • Synthesis of Disulfide Cross‐Linked DNAs Using Convertible Nucleosides
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Aboul‐ela, F., Koh, D., Tinoco, I. Jr., and Martin, F.H. 1985. Base‐base mismatches. Thermodynamics of double‐helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucl. Acids Res. 13:4811‐4824.
   Amaratunga, M., Snowden‐Ifft, E., Wemmer, D.E., and Benight, A.S. 1992. Studies of DNA dumbbells. II. Construction and characterization of DNA dumbbells with a 16 base‐pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14). Biopolymers 32:865‐879.
   Ashley, G.W. and Kushlan, D.M. 1991. Chemical synthesis of oligodeoxynucleotide dumbbells. Biochemistry 30:2927‐2933.
   Baldwin, R.L. 1971. Experimental tests of the theory of deoxyribonucleic acid melting with d(T‐A) oligomers. Acc. Chem. Res. 4:265‐272.
   Bannwarth, W., Dorn, A., Iaiza, P., and Pannekouke, X. 1994. Short optimally capped duplex DNA as a conformationally restricted analog of B‐DNA. Helv. Chim. Acta 77:182‐193.
   Benight, A.S., Schurr, J.M., Flynn, P.F., Reid, B.R., and Wemmer, D.E. 1988. Melting of a self‐complementary minicircle. Comparison of optical melting theory with exchange broadening of the nuclear magnetic resonance spectrum. J. Mol. Biol. 200:377‐399.
   Blatt, N.B., Osborne, S.E., Cain, R.J., and Glick, G.D. 1993. Conformational studies from the ColE1 cruciform. Biochimie 75:433.
   Boger, D.L., Munk, S.A., and Ishizaki, T. 1991. (+)‐CC‐1065 DNA alkylation: Observation of an unexpected relationship between cyclopropane electrophile reactivity and the intensity of DNA alkylation. J. Am. Chem. Soc. 113:2779‐2780.
   Borowy‐Borowski, H., Lipman, R., and Tomasz, M. 1990. Recognition between mitomycin C and specific DNA sequences for cross‐link formation. Biochemistry 29:2999‐3006.
   Breslauer, K.J. 1986. Thermodynamics of nucleic acids. In Thermodynamic Data for Biochemistry and Biotechnology (H.J. Hinz, ed.) pp 402‐427. Springer‐Verlag, New York.
   Breslauer, K.J., Frank, R., Blöcker, H., and Marky, L.A. 1986. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83:3746‐3750.
   Cain, R.J. and Glick, G.D. 1997. The effect of cross‐links on the conformational dynamics of duplex DNA. Nucl. Acids Res. 25:836‐842.
   Cain, R.J. and Glick, G.D. 1998. Use of cross‐links to study the conformational dynamics of triplex DNA. Biochemistry 37:1456‐1464.
   Cain, R.J., Zuiderweg, E.R.P., and Glick, G.D. 1995. Solution structure of a DNA hairpin and its disulfide cross‐linked analog. Nucl. Acids Res. 23:2153‐2160.
   Callahan, D.E., Trapane, T.L., Miller, P.S., Ts'o, P.O.P., and Kan, L.‐S. 1991. Comparative circular dichroism and fluorescence studies of oligodeoxyribonucleotide and oligodeoxyribonucleoside methylphosphonate pyrimidine strands in duplex and triplex formation. Biochemistry 30:1650‐1655.
   Catalano, C.E. and Benkovic, S.J. 1989. Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2′,3′‐epoxide 5′‐triphosphate: Evidence for the formation of a tight‐binding inhibitor. Biochemistry 28:4374‐4382.
   Clarke, J. and Fersht, A.R. 1993. Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation. Biochemistry 32:4322‐4329.
   Cowart, M. and Benkovic, S.J. 1991. A novel combined chemical‐enzymic synthesis of crosslinked DNA using a nucleoside triphosphate analog. Biochemistry 30:788‐796.
   Cowart, M., Gibson, K.J., Allen, D.J., and Benkovic, S.J. 1989. DNA substrate structural requirements for the exonuclease and polymerase activities of prokaryotic and phage DNA polymerases. Biochemistry 28:1975‐1983.
   Doktycz, M.J., Goldstein, R.F., Paner, T.M., Gallo, F.J., and Benight, A.S. 1992. Studies of DNA dumbbells. I. Melting curves of 17 DNA dumbbells with different duplex stem sequences linked by T4 endloops: Evaluation of the nearest‐neighbor stacking interactions in DNA. Biopolymers 32:849‐864.
   Durand, M., Chevrie, K., Chassignol, M., Thuong, N.T., and Maurizot, J.C. 1990. Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: Conformation and stability. Nucl. Acids Res. 18:6353‐6359.
   Elson, E.L., Scheffler, I.E., and Baldwin, R.L. 1970. Helix formation by d(TA) oligomers. 3. Electrostatic effects. J. Mol. Biol. 54:401‐415.
   Erie, D.A., Sinha, N., Olson, W.K., Jones, R.A., and Breslauer, K.J. 1987. A dumbbell‐shaped double‐hairpin structure of DNA: A thermodynamic investigation. Biochemistry 26:7150‐7159.
   Erie, D.A., Sinha, N.K., Olson, W.K., Jones, R.A., and Breslauer, K.J. 1989. Melting behavior of covalently closed, single‐stranded, circular DNA. Biochemistry 28:268‐273.
   Erlanson, D.A., Chen, L., and Verdine, G.L. 1993. DNA methylation through a locally unpaired intermediate. J. Am. Chem. Soc. 115:12583‐12584.
   Erlanson, D.A., Wolfe, S.A., Chen, L., and Verdine, G.L. 1997. Selective base‐pair destabilization enhances binding of a DNA methyltransferase. Tetrahedron 53:12041‐12056.
   Ferentz, A.E. and Verdine, G.L. 1991. Disulfide‐cross‐linked oligonucleotides. J. Am. Chem. Soc. 113:4000‐4002.
   Ferentz, A.E., Keating, T.A., and Verdine, G.L. 1993. Synthesis and characterization of disulfide cross‐linked oligonucleotides. J. Am. Chem. Soc. 115:9006‐9014.
   Frank‐Kamenetskii, M.D. 1985. Flunctuational motility of DNA. In Structure & Motion: Membranes, Nucleic Acids, and Proteins (E. Clementi, G. Corongiu, M.H. Sarma, and R.H. Sarma, eds.) pp. 417‐432. Adenine Press, Guilderland, N.Y.
   Freier, S.M., Kierzek, R., Caruthers, M.H., Neilson, T., and Turner, D.H. 1986. Free energy contributions of G·U and other terminal mismatches to helix stability. Biochemistry 25:3209‐3213.
   Gao, H., Chidambaram, N., Chen, B.C., Pelham, D.E., Patel, R., Yang, M., Zhou, L., Cook, A., and Cohen, J.S. 1994. Double‐stranded cyclic oligonucleotides with non‐nucleotide bridges. Bioconjugate Chem. 5:445‐453.
   Gao, H., Yang, M., and Cook, A.F. 1995. Stabilization of double‐stranded oligonucleotides using backbone‐linked disulfide bridges. Nucl. Acids Res. 23:285‐292.
   Germann, M.W., Schoenwaelder, K.‐H., and van de Sande, J.H. 1985. Right‐ and left‐handed (Z) helical conformations of the hairpin d(C‐G)5T4(C‐G)5 monomer and dimer. Biochemistry 24:5698‐5702.
   Glick, G.D. 1991. Synthesis of a conformationally restricted DNA hairpin. J. Org. Chem. 56:6746‐6747.
   Glick, G.D. 1998. Design, synthesis, and analysis of conformationally constrained nucleic acids. Biopolymers 48:83‐96.
   Glick, G.D., Osborne, S.E., Knitt, D.S., and Marino, J.P. Jr. 1992. Trapping and isolation of an alternate DNA conformation. J. Am. Chem. Soc. 114:5447‐5448.
   Goodwin, J.T. and Glick, G.D. 1993. Incorporation of alkylthiol chains at C‐5 of deoxyuridine Tetrahedron Lett. 34:5549‐5552.
   Goodwin, J.T., Osborne, S.E., Swanson, P.C., and Glick, G.D. 1994. Synthesis of a disulfide cross‐linked DNA triple helix. Tetrahedron Lett. 35:4527‐4531.
   Guéron, M. and Leroy, J.L. 1995. Studies of base pair kinetics by NMR measurement of proton exchange. Methods Enzymol. 261:383‐413.
   Haasnoot, C.A.G., Hilbers, C.W., van der Marel, G.A., van Boom, J.H., Singh, U.C., Pattabiraman, N., and Kollman, P.A. 1986. On loop folding in nucleic acid hairpin‐type structures. J. Biomol. Struct. Dyn. 3:843‐857.
   Häner, R. and Dervan, P.B. 1990. Single‐stranded DNA triple‐helix formation. Biochemistry 29:9761‐9765.
   Kiessling, L.L., Griffin, L.C., and Dervan, P.B. 1992. Flanking sequence effects within the pyrimidine triple‐helix motif characterized by affinity cleaving. Biochemistry 31:2829‐2834.
   Kirchner, J.J. and Hopkins, P.B. 1991. Nitrous acid cross‐links duplex DNA fragments through deoxyguanosine residues at the sequence 5′‐CG. J. Am. Chem. Soc. 113:4681‐4682.
   Kirchner, J.J., Sigurdsson, S.T., and Hopkins, P.B. 1992. Interstrand cross‐linking of duplex DNA by nitrous acid: Covalent structure of the dG‐to‐dG cross‐link at the sequence 5′‐CG. J. Am. Chem. Soc. 114:4021‐4027.
   Kool, E.T. 1991. Molecular recognition by circular oligonucleotides: Increasing the selectivity of DNA binding. J. Am. Chem. Soc. 113:6265‐6266.
   Lemaire, M.‐A., Schwartz, A., Rahmouni, A.R., and Leng, M. 1991. Interstrand cross‐links are preferentially formed at the d(GC) sites in the reaction between cis‐diamminedichloroplatinum(II) and DNA. Proc. Natl. Acad. Sci. U.S.A. 88:1982‐1985.
   MacGregor, R.B. Jr. 1996. Chain length and oligonucleotide stability at high pressure. Biopolymers 38:321‐327.
   Marky, L.A., Blumenfeld, K.S., Kozlowski, S., and Breslauer, K.J. 1983. Salt‐dependent conformational transitions in the self‐complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation. Biopolymers 22:1247‐1257.
   Millard, J.T., Raucher, S., and Hopkins, P.B. 1990. Mechlorethamine cross‐links deoxyguanosine residues at 5′‐GNC sequences in duplex DNA fragments. J. Am. Chem. Soc. 112:2459‐2460.
   Millard, J.T., Weidner, M.F., Kirchner, J.J., Ribeiro, S., and Hopkins, P.B. 1991. Sequence preferences of DNA interstrand crosslinking agents: Quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites. Nucl. Acids Res. 19:1885‐1891.
   Moser, H.E. and Dervan, P.B. 1987. Sequence‐specific cleavage of double helical DNA by triple helix formation. Science 238:645‐650.
   Olmsted, M.C., Anderson, C.F., and Record, M.T. Jr. 1991. Importance of oligoelectrolyte end effects for the thermodynamics of conformational transitions of nucleic acid oligomers: A grand canonical Monte Carlo analysis. Biopolymers 31:1593‐1604.
   Osborne, S.E. 1996. Ph.D. Thesis, University of Michigan.
   Osborne, S.E., Völker, J., Stevens, S.Y., Breslauer, K.J., and Glick, G.D. 1996. Design, synthesis, and analysis of disulfide cross‐linked DNA duplexes. J. Am. Chem. Soc. 118:11993‐12003.
   Osborne, S.E., Cain, R.J., and Glick, G.D. 1997. Structure and dynamics of disulfide cross‐linked DNA triple helices. J. Am. Chem. Soc. 119:1171‐1182.
   Paner, T.M., Amaratunga, M., and Benight, A.S. 1992. Studies of DNA dumbbells. III. Theoretical analysis of optical melting curves of dumbbells with a 16 base‐pair duplex stem and Tn end loops (n = 2, 3, 4, 6, 8, 10, 14). Biopolymers 32:881‐892.
   Patel, D.J. and Hilbers, C.W. 1975. Proton nuclear magnetic resonance investigations in double‐stranded dApTpGpCpApT in aqueous solution. Biochemistry 14:2651‐2656.
   Pilch, D.S., Levenson, C., and Shafer, R.H. 1990. Structural analysis of the d(A)10.2(dT)10 triple helix. Proc. Natl. Acad. Sci. U.S.A. 87:1942‐1946.
   Pinto, A.L. and Lippard, S. 1985. Binding of the antitumor drug cis‐diamminedichloroplatinum(II) (cisplatin) to DNA. Biochem. Biophys. Acta 780:167‐180.
   Plum, G.E., Pilch, D.S., Singleton, S.F., and Breslauer, K.J. 1995. Nucleic acid hybridization: Triplex stability and energetics. Annu. Rev. Biophys. Biomol. Struct. 24:319‐350.
   Ramstein, J. and Lavery, R. 1988. Energetic coupling between DNA bending and base pair opening. Proc. Natl. Acad. Sci. U.S.A. 85:7231‐7235.
   Ramstein, J. and Lavery, R. 1990. Base pair opening pathways in B‐DNA. J. Biomol. Struct. Dyn. 7:915‐933.
   Record, M.T. Jr. and Lohman, T.M. 1978. Semi‐empirical extension of polyelectrolyte theory to treatment of oligoelectrolytes—application to oligonucleotide helix‐coil transitions. Biopolymers 17:159‐166.
   Rumney, S. IV and Kool, E.T. 1992. DNA recognition by hybrid oligoether‐oligodeoxynucleotide macrocycles. Angew. Chem. Int. Ed. Engl. 31:1617‐1619.
   Salunkhe, M., Wu, T., and Lestinger, R.L. 1992. Control of folding and binding of oligonucleotides by use of a nonnucleotide linker. J. Am. Chem. Soc. 114:8768‐8772.
   SantaLucia, J. Jr., Allawi, H.T., and Seneviratne, P.A. 1996. Improved nearest‐neighbor parameters for predicting DNA duplex stability. Biochemistry 35:3555‐3562.
   Scheffler, I.E., Elson, E.L., and Baldwin, R.L. 1970. Helix formation by d(TA) oligomers. II. Analysis of the helix‐coil transitions of linear and circular oligomers. J. Mol. Biol. 48:145‐171.
   Sherman, S.E., Gibson, D., Wang, A.H., and Lippard, S.J. 1985. X‐ray structure of the major adduct of the anticancer drug cisplatin with DNA: cis‐[Pt(NH3)2] (d(pGpG)). Science 230:412‐417.
   Sigurdsson, S.T., Rink, S.M., and Hopkins, P.B. 1993. Affinity crosslinking of duplex DNA by a pyrrole‐oligopeptide conjugate. J. Am. Chem. Soc. 115:12633‐12634.
   Singleton, S.F. and Dervan, P.B. 1992. Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide‐directed triple helix formation at single DNA sites. Biochemistry 31:10995‐11003.
   Stevens, S.Y., Swanson, P.C., Voss, E.W. Jr., and Glick, G.D. 1993. Evidence for induced fit in antibody·DNA complexes. J. Am. Chem. Soc. 115:1585‐1586.
   Sun, J.‐S. and Hélène, C. 1993. Oligonucleotide‐directed triple‐helix formation. Curr. Opin. Struct. Biol. 3:345‐356.
   Swanson, P.C., Cooper, B.C., and Glick, G.D. 1994. High‐resolution epitope mapping of an anti‐DNA autoantibody using model DNA ligands. J. Immunol. 152:2601‐2612.
   Tari, L.W. and Secco, A.S. 1995. Base‐pair opening and spermine binding—B‐DNA features displayed in the crystal structure of a gal operon fragment: Implications for protein‐DNA recognition. Nucl. Acids Res. 23:2065‐2073.
   Teng, S.P., Woodson, S.A., and Crothers, D.M. 1989. DNA sequence specificity of mitomycin cross‐linking. Biochemistry 28:3901‐3907.
   Thuong, N.T. and Hélè, C. 1993. Sequence‐specific recognition and modification of double‐helical DNA by oligonucleotides. Angew. Chem. Int. Ed. Engl. 32:666‐690.
   Tomasz, M., Lipman, R., Chowdary, D., Pawlak, J., Verdine, G.L., and Nakanishi, K. 1987. Isolation and structure of a covalent cross‐link adduct between mitomycin C and DNA. Science 235:1204‐1208.
   Völker, J., Osborne, S.E., Glick, G.D., and Breslauer, K.J. 1997. Thermodynamic properties of a conformationally constrained intramolecular DNA triple helix. Biochemistry 36:756‐767.
   Wang, H., Osborne, S.E., Zuiderweg, E.R.P., and Glick, G.D. 1994. Three‐dimensional structure of a disulfide‐stabilized non‐ground‐state DNA hairpin. J. Am. Chem. Soc. 116:5021‐5022.
   Wang, H., Zuiderweg, E.R.P., and Glick, G.D. 1995. Solution structure of a disulfide cross‐linked DNA hairpin. J. Am. Chem. Soc. 117:2981‐2991.
   Webb, T.R. and Matteucci, M.D. 1986. Sequence‐specific cross‐linking of deoxyoligonucleotides via hybridization‐triggered alkylation. J. Am. Chem. Soc. 108:2764‐2765.
   Wemmer, D.E. and Benight, A.S. 1985. Preparation and melting of single‐strand circular DNA loops. Nucl. Acids Res. 13:8611‐8621.
   Williams, D.J. and Hall, K.B. 1996. Thermodynamic comparison of the salt dependence of natural RNA hairpins and RNA hairpins with non‐nucleotide spacers. Biochemistry 35:14665‐14670.
   Willis, M.C., Hicke, B.J., Uhlenbeck, O.C., Cech, T.R., and Koch, T.H. 1993. Photocrosslinking of 5‐iodouracil‐substituted RNA and DNA to proteins. Science 262:1255‐1257.
   Wing, R., Drew, H., Takano, T., Broka, C., Tanaka, S., Itakura, K., and Dickerson, R.E. 1980. Crystal structure analysis of a complete turn of B‐DNA. Nature 287:755‐758.
   Wolfe, S.A. and Verdine, G.L. 1993. Ratcheting torsional stress in duplex DNA. J. Am. Chem. Soc. 115:12585‐12586.
   Wolfe, S.A., Ferentz, A.E., Grantcharova, V., Churchill, M.E.A., and Verdine, G.L. 1995. Modifying the helical structure of DNA by design: Recruitment of an architecture‐specific protein to an enforced DNA bend. Chem. Biol. 2:213‐221.
PDF or HTML at Wiley Online Library