J. Am. Chem. Soc. 1994,116, 785-786
Cooperative Binding of 8-mer Oligonucleotides Containing 5-( l-Propynyl)-2’-deoxyuridineto Adjacent DNA Sites by Triple-Helix Formation
785
h
. . . . . . . . . . . . . . .3’ . .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
3’
3 ’ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5’ 8bpSleA
Natalia Colocci and Peter B. Dervan’
8bpSiteB
5‘-A A A
AGAJ’
Division of Chemistry and Chemical Engineering Cali/ornia Insrifute of Technology Pasadena, California 91 125
3’-T T T
TCT-5’
1
S-’TUCCUUUU
2 S-UUUUCUCT
Received October 18. 1993
3
s-*nEm
4
The design of cooperative domains between DNA-binding ligands for modulation of the kinetics and thermodynamics of ligand-nucleic acid interactions is a t an early stage of development.‘+ Pyrimidine oligdeoxyribonucleotides 1 1 nucleotides (nt) in length are known to bind cooperatively to abutting sites on double-helical D N A by triple-helix formation, resulting in a 20-fold increase in the association constant for one oligonucleotide in the presence of the neighboring oligonucleotide.’ This 1.8 kcalmol-I interaction energy may arise from *-stacking between the bases a t the triple-helical junction.’ The incorporation of discrete dimerization domains to oligonucleotides such as those capable of forming a short Watson-Crick helix produces a cooperative interaction and results in a 44-fold enhancement in the association constant of one oligonucleotide in the presence of a neighbor? An alternative approach to increasing cooperativity by the addition of dimerization domains is the use of modified bases, whichcouldallow greater interactionsbetween basesat thetriplehelical junction. It is known that replacing 2‘-deoxycytidines in the third strand with S-methyl-2’-deoxycytidines, and replacing 2‘-deox yuridines with thymidines, S-ethynyl-2‘-deoxyuridines,or 5-( 1-propynyl)-2’-deoxyuridines,increases the stability of triplehelical complexes.s.6 Ifthis enhancedstability is due to increased stacking in the third strand, one might expect similar effects a t triple-helix junctions. W e report here that two short oligodeoxyribonucleotides containing S-(l-propynyl)-substituted 2’deoxyuridine (PU) and 5-methyl-2’-deoxycytidine ( M C ) bind cooperatively to adjacent sites on double-helical D N A a t micromolar concentrations (IO mM Bis-Tris-HCI at pH 7.0, 10 mM NaC1,I mM spermine, 24OC) (Figure 1). Theequilibrium constant of an 8-mer binding in the presence of a neighboring bound %mer is enhanced by a factor of at least 40 (relative to the 8-mer alone) under the conditions used. A system was designed in which two 8-nt oligodeoxyribonucleotides, 1 and 2, bind site-specifically to adjacent sites on a n 850-bp 3’-”P-end-labeled duplex D N A fragment via specific Hoogsteen hydrogen bonding (PU-AT and MeC GC) (Figure
+
( I ) Strobel, S.A.; LkNan. P. B. J. Am. Chem. Soe. 1989, 111, 7286. (2) (21 (a) Distcfano, M. D.: D.; Shin. Shin, J. A,: A,; DcN~”. DcN~”,P. B. J . Am. Chem. Soe. , . la) . , Distcfano. 1991, 113, 5901. (b) Dirtcfano. Dirtefano. M. M. 0.: D.; D cc~~a na P. ,i B. J . Am. Chem. Soe. 1992,114,11006. (c) ( c ) Distcfano,M. D.;Dcrvan, P. B.Proe. Notl. Aeod.Sci. U.S.A. 1993. 90, 1179. i3) Colacci.. N.:. Distefano. M. D.: DeNan. P. 8.J . Am. Chem. Soe. 1993. ilj, 4468. (4) Mergny, J. L.; Duval-Valcntin. G.; Nguycn, C. H.;Perrauault, L.: Faumn. B.; Roup&, M.: Montenay-Garesticr, T.; Biaagni, E.;Helkne, C. Science 1992. 256. 1681. (5) (a) Lee, J. S.; Wwdsworth. M. L.; Latimer. L. J. P.; Morgan, A. R. Nucleic Acids Res. 1984. 12,6603. (b) Povsic, T. J.; Dervan, P. B. 3. Am. Chrm. Soe. 1989.111.3059. (e) Povsic, T. J. Ph.D. Dissertation, California InstituteofTechnalopv. Pasadena. CA. 1991. (d) Collier. D. A,: Thuane. N. (6) (a) Fraehler. B. C ; Wadwan,. S.:Tcrhorst. T. J.: Gcrrard. S . R. TetrahedronLeu 1992.33, 5301. For 1hccffcn of 5-( I -propynyl)p)rimidincs on duplex stability. see: ( b ) Sagi. J.: Szcmzo. A : Ebingcr. K.. Subolcr. A.: Sag,. ( i . ; Rufl. E..Otvos. L.: Tetrahedron Lett. 1993. 34. 2191
eu.”.PP
....
P P
YIY
S-TTITZTZT
-w e
“W b
-,Lo
-eP-O
. . A U L C Fipre 1. Schematic representation of a complex wmposcd of two triplo-
helix-forming oligonucleotides binding at adjacent sites on double-helical DNA. Thick solid lines represent the DNA backbone of the target site and associated oligonucleotides. Thin solid lines represent Watson-Crick hydrogen bonds while dashed lines indicate Hwgsteen hydrogen bonds. Binding of the oligonucleotides is assessed by affinity cleavage using T’. PU and MeC represent the C5-propynyl-substituted2’-deoxyuridine and 5-rnethyl-Z’-deoxycytidine nucleotides, respectively l).’ ThymidineEDTA (T’) was incorporated at the 5’-terminus of oligonucleotide 1 to allow thermodynamic analysis of sitespecific binding using the quantitative affinity cleavage titration method?JO To determine the interaction energy between oligonucleotides 1 and 2, the equilibrium association constants K, for 1 binding to site A alone and Kt.2 for 1 binding to site A in the presence of 1.0 pM 2 bound to neighboring site B were measured.’J0 Analysis of cleavage data yielded equilibrium association constants of I 40 (Table This corresponds to a cooperative interaction energy of >4.5 kcalmol-1.12 For comparison, binding of the corresponding unmodified oligonucleotides 3 and 4containinga methyl (thymidine) instead of a propynyl group a t the 5-position of Z’-deoxyuridine, was analyzed under the same conditions (Figure I). Oligonucleotide 3 a t 5100rMbothintheabsenccandinthepresenceof 1 . 0 p M
.. ~.
&PI& the S.DMT.pkX&d ;.I I-propyn)lJ-2’-dmiyurldlne’lo mntrdl porn glass support using standard methodology.’ Ohgonuelmtides I and 2 vcrerubpld tostandardcn2)mcdegradation and IlPLC analysis lor purtty
confirmation. (8) Gait, M. J., Ed. Oligonucleotide Synthesis: A Proclicol Approach: IRL Press: Oxford. 1984. (9) Dreyer. G. B.; Dewan, P. 8.Proe. Notl. Acod. Sei. U.S.A. 1985.82, 968.
(lO)(a)Singleton,S.F.;Dervan,P.B.J.Am.Chem.Soe.1992,111,6957. (b) Singleton, S.F.;Dervan, P. 6. Biochemistry 1992, 31, 10995. (,I) I (a) Under the conditions chosen. oligonucleotide I alone at mncentrations up to 100 pM produced only minimal cleavage. It was therefore assumed that K I is
