A theoretical study of the interaction of 4′,6 diamidino-2-phenylindole (DAPI) with the double-stranded oligonucleotides (dA-dT)11 and (dA)11·(dT)11

A theoretical study of the interaction of 4',6 diamidino-2-phenylindole (DAPI) with the double-stranded oligonucleotides (dA-dT)ll and (dA)ll.(dT)ll Nohad Gresh lnstitut de Biologie Physico-Chimique, Laboratoire de Biochimie Th~orique, associe au CNRS, 13, rue Pierre et Marie Curie, 75005 Paris, France (Received 15 January 1985; revised 26 March 1985) The nonintercalative binding of DAPI to the minor groove of double-stranded (dA--dTht and (dA)ll.(dT)11 oligomers held in the B-DNA conformation is investigated by performing theoretical computations of related intermolecular interaction energies. For both oligomers, the intrinsically preferred binding configuration is stabilized by hydrogen bonding interactions involving side A of D API and 02 and N 3 atoms belonging to the (Y3') strand of (dA-dT)l 1 or 0 2 of the thymine strand of (dA)l I . (dT)l 1. Additional interactions involve hydrogen atoms of side B of DAPI and Or, deoxyribose oxygens of the opposite strand.

Keywords: DNA; DAPI; double-stranded oligonucleotides

Introduction The diarylamidine 4',6 diamidino-2-phenylindole (DAPI, Figure 1), synthesized in 19711, has been widely utilized in studies of chromosome banding 2- 6, as well as to probe the molecular environment of restriction endonuclease cleavage sites 7. Its interaction with D N A or synthetic polynucleotides has lent itself to several physicochemical studiesa_13. A distinct preference for (A-T) rich sequences of DNA rather than (G-C) rich sequences was evidenced 8,12,13. The preferred binding configuration of DAPI to DNA remains unkown. Both intercalative a'l 1,12 and nonintercalative 1°'13 binding modes were advocated to occur. The recent results of reference 13, which are in support ofa nonintercalative binding, have motivated the present study, in which a search is undertaken of the intrinsically preferred nonintercalative binding configuration(s) of DAPI in the minor groove of DNA, by performing theoretical computations of its intermolecular interaction energy with this groove. This work is a continuation of previous theoretical studies of our laboratory, devoted to nonintercalative cationic DNA binding compounds, and performed on netropsin and SN 1807114, bisguanylhydrazones 15, and the diarylamidines berenil and stilbamidine 16. The preferential affinity of this type of compound 17 for the minor groove of A-T sequences with respect to the minor groove of G-C sequences or the major groove of the two types of sequences having been previously exemplified on the related examples of netropsin and SN 1807114, we will investigate here and optimize only the binding of DAPI to the minor grove of a double helix built out of an 0141-8130/85/0401994)4503.00 © 1985 Butterworth & Co. (Publishers) Ltd

undecanucleoside decaphosphate with either alternating or nonalternating sequences (Figure 2). This search is performed first for naked DNAs with unscreened phosphates. The effect of countercation binding to the phosphates is further exposed, by linking a monovalent cation to each phosphate in a configuration bridging the two anionic oxygens ~s, as performed previously 14- x6

Procedure The intermolecular interaction energies are computed by means of an additive procedure, elaborated previously in this laboratory 19, and applied to a number of problems related to binding specificities, including nonintercalative DNA binding compounds 15,16 or cation-ionophore interactions 2° (see reference 19 for details). It was shown to reproduce satisfactorily the results of ab initio SCF supermolecule computations in representative cases ~9,2~ or experimental results when available 22.2a.

SideA

78' H~' H8 ' "T +N - '

~

Y2' ~

.

HI \

~7

i

~!I+ '. NGH~ z 2

,_.,/ H4. SideB

Figure 1 DAPI, structural formula and atom numbering

Int. J. Biol. Macromol., 1985, Vol 7, August

199

Interaction of DAPI with double-stranded oligonucleotides: N. Gresh

3'

5 I

~5

A .S . . . . .

P-4:

~5 P -4'

#5

P

r- 5 P

~5--

T÷3~5.. ~ F

A.2~5

T+2 . . . . .

~

P.,a< .~,.5 ~ P'-4:_ .~.5-

T o .....

A o ~5.,,~

A_~ . . . . .

T_ ~

T- 2. . . . .

A_2 ~ 5 " ~

5

~5-P -4' s~..5-

-

5~

A+s ..... T + s ~ 5 . , ~

.....

-

T_,

.

.

.

r_,

.

~,,5P

~"P

5fi-~'P

S'

T+a--5~

A+z .....

T +2--5~

A+, . . . . .

T+, - - 5 " ~

A o .....

T o--5, 4 _

.¢,v

P"f': 5 - 3'

a

A- 2 . . . . .

,~,

.

P " ~ 5 . . A_. s -. . . T_ s

A+3 . . . . .

P,<

P

P"~':5

T-2 ~5~,,~, P _

,...~,,, I-'

A_ 4 . . . . .

T_ 4 - - 5,,,,{, p,~

A-s .....

T_ s - - S

S'

,

b

2 (a) (b), double-stranded (dAviT h ~ and (dAh 1. (dTh 1. Notations adopted for the constituent strands

The interaction energy is computed as a sum of four components: EMT P "4-Epol+

E,~ + E,ll

where EMTp,E0otand Erep a r e the electrostatic, polarization and repulsion terms respectively, and Ed~is dispersion-like term which includes the charge-transfer when present. The electrostatic multipolar term EMTp,which brings about the major contribution to the binding energy, is computed as a sum of multipole-multipole interactions, by using an overlap multipole expansion of the ab initio SCF wave functions of the constituent fragments of DNA and of that of DAPI. This expansion locates multipoles (up to quadrupoles) on all the atoms and all the bond barycentres of the interacting molecules. To compute the polarization term, we use experimental polarizabilities z4, partitioned on the atoms and the bond barycentres, whereas the polarizing field is computed by means of a multipolar expansion. The repulsion term is computed as a sum of bond-bond repulsions. The calibration of E0o~, E,cp and Ed~ was done originally in an earlier work 19. Let us recall that the values of the constants D and F, which appear in the denominators of E~,t and Edi respectively, are 0.45 and 0.015; the value of 0t, which appears in the exponential terms of E,ep, is 12.35 and the values of A and C, which are the multiplicative constants of E,, and E,cp respectively, are 0.214 and 30322, all distances being expressed in Angstrtim units and the energies in kcal/mol. The effective radii on H, Caliphatic, Caromatic, N, O and P are 1.2, 1.7, 1.77, 1.7, 1.5 and 1.85 respectively. The nucleic acid oligomers were constructed from their constituent fragments in the same fashion as that adopted for the computation of the molecular electrostatic

200

4.'

P i

AE =

--

P , A+3 . . . . .

Figure

5'

31

T +5 ~ 5 ~

Int. J. Biol. Macromol., 1985, Vol 7, August

potential of large macromolecules 25, the fragments being the two bases, deoxyribose and methylphosphate. The basis set used in the ab initio computations of the fragments is the one given in reference 18. The wave function of DAPI was computed using the Melius-Topiol pseudopotential procedure 26 using the basis set discussed in reference 27. We have retained throughout this study the standard B-DNA conformation, as given by the refined coordinates published by Arnott et al. in 198028. This choice was adopted on account of the results of recent theoretical computations of the proton shifts of the bases in a model dodecamer 29, which indicate the relevance of these coordinates to situations in solution. The choice of a regular B-DNA conformation rather than an alternating B-DNA conformation 3° for the alternating sequence is further supported by the results of recent N M R Nuclear Overhauser Enhancement measurements performed on poly(dA-dT) 31. A standard internal geometry was used for DAP132. Let A, B and C denote three successive linked atoms on the oligonucleotide and H: 1, Nx x and HI 1 the first three linked atoms of DAPI. The configuration of approach of DAPI to the oligonucleotide is then defined by a set of six intermolecular variables, namely: one intermolecular distance Ht1...A; two angles: H t x . . . A - B and N11-H11-.. A; and three dihedral angles, H t l . . . A-B-C, Nil-H11... A-B, and H~ 1-N11-H11... A. The procedure adopted conforms itself to an algorithm derived by Thomson 33. The search for the optimal binding configuration of DAPI in the minor groove is performed by means of an energy-minimization procedure using Fletcher's algorithm 34, the interaction energy being optimized as a function of the six intermolecular variables,

Interaction of DAPI with double-stranded oli#onucleotides: N. Gresh Table I Values of the optimized interaction energies of DAPI with double-stranded oligomers (dA-dT h i and (dAh 1- (dTh i. Energies in kcal/mol (see text for definitions) Unscreened phosphates (dA~IT)tl (dA)lt.(dT)ll AE

-1194.5 -1126.0 EpoI -39.1 E~p +59.9 gdl - - 89.4 EMTP

-1197.1 -1125.6 -38.3 +51.2 - 84.4

and the three dihedral angles defming the conformation or DAPI proper. A large number of such minimizations were performed by selecting different starting points, so as to ensure that configurations of interest will not be overlooked. We will present here the results pertaining to the so-derived global energy minima. Several configurations closely related to them, both energetically and geometrically, were also derived (unpublished, available upon request).

Screened phosphates (dA~lT)ll (dA)ii.(dT)ll -272.2 -204.0 -38.7 +59.9 - 89.4

-276.0 -204.9 -37.9 +51.2 - 84.4

Results and discussion The interaction energies of DAPI with the two oligomers are reported in Table 1, both at the unscreened and the screened phosphate levels, together with the values of the energy contributions to the binding. Table 2 reports the values of the most significant hydrogen bond distances between heteroatoms of the minor groove (02, N3 and Or) and DAPI. The intrinsically preferred binding configuration of DAPI is such that hydrogen atoms belonging to side A of the molecule, namely atoms H tt , H 7 , H I and H 3, interact with three bases oftbe 5'3' strand of the alternating oligomer, namely 0 2 (T+2), Na(A + 1) and O2(To) , and atoms 0 2 ofT+ 2, T+ l and T o of the thymine strand of the homo-oligomer. Additional interactions occur between Or, deoxyribose oxygens belonging to the opposite strand and hydrogen atoms of DAPI of side B, namely Ha, H6,, Hv, and H 9, (see Table 2). With respect to the all-planar conformation of DAPI,

Table 2 Values of the optimized interatomic distances between DAPI and atoms of the minor groove. (Distances in A) (dA-dT)x 1

(dA)x 1. (dT)l 1

H I I-O2(T+2) 1.82} H7-Oz(T+ 2) 2.06[ 5'3' strand H1-N3(A+ l) 1.96 H3,~D2(To) 2.24J

H 11-O2(T- 2) 2.00I H7-O2(T_ 2) 2.13/ Thymine H1-O2(T- l) 1.73} strand H2,-O2(T- l) 2.15/ Hy~)2(T0) 2.1.9!

H3-OI,(S_ 1) 1.82] H6,~:)t'(S- I) 2.28 1`3'5' Hs,-OI,(S_2) 2.15 [ strand H9,-OI,(S_ 2) 1.891

H3-O1,(8+ 1) 2.14} H6'--Ot'(S+ t) 2.121, Adenine Hs,--O I,(S+2 ) 2.181 strand H9,~OI,(S +2) 1.87J

T+ 2

~" ~

o

/-x

/'-%

I

A+2 •

T+I

¢-,

TO A0

A_ 1 ',

T_ 1

(-~ T_ 2

f

J

A_ 2

s

~,

/

x_,

Figure 3 Representation of the preferred binding mode of DAPI with double-stranded (dA-dT).

Int. J. Biol. Macromol., 1985, Vol 7, August

201

Interaction o f D A P I with double-stranded oligonucleotides: N. Gresh

very limited conformational changes occur along bonds C1o-C6, C2-Cr, and C4.,-C7,. The respective torsional angle variations are of - 20°, 10° and - 5° (respectively) in the alternating oligomer and - 7 °, - 7 ° and - 2 ° (respectively) in the homo-oligomer. Thus, the overall conformation of DAPI remains close to planar. The binding configuration of DAPI with the alternating oligomer is represented in Figure 3, drawn with the help of the Figatom program 35. For the sake of clarity, only the fragment of the oligomer with three base pairs on both sides of the central base pair is represented. As in reference 16, the predominant role of the electrostatic contribution, EMTp! 9, to the overall binding energy can be underlined, a reflection ol the strong attractive values of the molecular electrostatic potential computed in the minor groove of A-T sequences of B--DNA 36'37. The numerical values of the interaction energies of DAPI with the alternating oligomer are only slightly smaller than the ones derived with the homo-oligomer. It must be recalled, however, that the present investigation was performed with a fixed B-DNA conformation for both investigated sequences. Recent calorimetric measurements indicate, in fact, the affinities of DAPI for the two polynucleotides poly(dA-dT) and poly(dA). poly(dT) to be equal ~~. The theoretically established preferred binding model of DAPI to the undecanucleosides is compatible with the proposals put forward on the basis of experimentation ~3. The results of the present study are in line with those of previous studies, devoted to the binding to B-DNA of nonintercalating ligands 14-~6 and cations 3a, and the implications from studies of the molecular electrostatic potential of B--DNA for A-T sequences 36'37. They show that an adequate fitting between DAPI and the minor groove of (dA-dT)l i or (dA)11. (dTh 1 can be readily attained, and is translated by a large number of stabilizing interactions involving hydrogen atoms of DAPI and specific sites (02, O1,, N3) on up to five base pairs of the oligonucleotides. As previously underlined 14-16, the absolute values of the gas-phase interaction energy derived in this study should not be correlated quantitatively with experimental data on the complexation enthalpies of DAPI by the oligonucleotide in solution ~a. Such a correlation would require a more exhaustive inclusion of the solution environment of the complex. This inclusion was was carried out in a recent study of our laboratory a9, devoted to other nonintercalating ligands. It was shown to result in a reduction of the overall interaction energy, to values close to those of available experimental determinations. We did not implement such a treatment in the present study, since, in keeping with the conclusions reached in reference 39, it is not likely to alter the groove preference of nonintercalating dicationic ligands, nor their intrinsically preferred binding characteristics in the groove.

Acknowledgements The author wishes to thank Professor Bernard Pullman for critically reviewing the manuscript prior ro publication. The computations presented in this study

202 Int. J. Biol. Macromol., 1985, Vol 7, August

were performed on the VAX/750 computer of the National Foundation for Cancer Research. We are pleased to acknowledge the decisive help of NFCR which enabled the realization of the present project.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Dann, O., Bergen, G., Demant, E. and Volz, C., Justus Liebigs Annalen Chem. 1971, 749, 68 Russell, W., Newman, C. and Williamson, D. Nature 1975, 253, 461 Hajduk, S. Science, 1976, 191, 858 Schweizer, D. Exp. Cell. Res. 1976, 102, 408 Lin, M., Alfi,O. and Donnel, G. Can. J. Genet. Cytol. 1976,18, 545 Schweizer, D. Chromosoma, 1976, $8, 307 Kania, J. and Fanning, T. Eur. J. Biochem. 1976, 67, 367 Chandra, P., Mildner, B., Dann, O. and Metz, A. Mol. Cell. Biochemistry, 1977, 18, 81 Bierzynski, A., Boguta, G., Kerens, K. and Wierzchowski, K. Studia Biophys, 1978, 67, S 57 Stepien, E., Dann, O., Fikus, M. and Wierzchowski, K., Studia Biophys. 1978, 67, S 135 Kapuscinski, J. and Skocylas, B., Nucleic Acids Res. 1978,5, 3775 Kapuseinski, J. and Szer, W. Nucleic Acids Res. 1979, 6, 3519 Manzini, G., Bareellona, M., Avitabile, M. and Quadrifoglio, F. Nucleic Acids Res. 1983, I1, 8861 Zakrzewska, K., Lavery, R. and Pullman, B. Nucleic Acids Res.

1983, 24, 8825 Gresh,N. and Pullman, B. Theoret. Chim. Acta 1984,64, 383 Gresh,N. and Pullman, B. Mol. Pharmacol. 1984,25, 452 Baguley,B., Mol. Cell. Biochem. 1982, 43, 167 and references therein Pullman,B., Gresh, N., Berthod, H. and Pullman, A. Theoret. Chim. Acta 1977, 44, 151 Gresh, N., Claverle, P. and Pullman, A. Int. J. Quantum Chem. Symp. 1979, 13, 243 Gresh, N. and Pullman, A. Int. J. Quantum Chem., Quantum Biol. Symp. 1983, 10, 215 Gresh, N. and Pullman, B. Theoret. Chim. Acta 1979, 52, 67 Gresh, N. and Pullman, B. Biochim. Biophys. Acta 1980, 608, 47 Langlet, J., Claverle, P., Caron, F. and Boeuve, J. C. Int. J. Quantum Chem. 1981, 19, 299 Le Fevre, R. in 'Advances in Physical Organic Chemistry', 1965, 3,1 Pullman, A., Zakrzewska, K. and Perahia, D. Int. J. Quantum Chem. 1979, 15, 395 Topiol, S., Moskowitz, J. and Melius, C. J. Chem. Phys. 1978, 68, 2364 Gresh, N. and Pullman, A. Theoret. Chim. Acta 1978, 49, 283 Arnott, S., Chandrasekaran, R., Birdsall, D., Leslie, A. and Ratliff, R. Nature 1980, 283, 743 Giessner-Prettre, C. and Pullman, B. Biochem. Biophys. Res. Comm. 1982, 107, 1539 Klug, A., Jack, A. Viswamitra, M., Kennard, O., Shakked, Z. and Steitz, T. J. Mol. Biol. 1979, 131, 669 Assa-Munt, N. and Kearns, D. Biochemistry 1984, 23, 791 'Molecular structures and dimensions', vol. A 1, Crystallographic Data Center, University Chemical Laboratory, Cambridge and International Union of Crystallography, 1972 Thomson, H. J. Chem. Phys. 1967, 47, 3407 Fletcher, R. 'Fortran Subroutines for Minimization by QuasiNewton Methods', AERE Report R7125, 1972 Langlet, G. J. Appl. Crystallogr. 1972, 5, 66 Pullman, B. and Pullman, A. Studia Biophys. 1981, 86, 95 Pullman, B., Pullman, A. and Lavery, R. in 'Structure, Dynamics, Interactions and Evolution of Biological Macromolecules', (Ed. C. Helene), 1983, p. 23 Gresh, N. and Pullman, B. Int. J. Quantum Chem. 1983, 24, 491 Zakrzewska, K., Lavery, R. and Pullman, B. Nucleic Acids Res. 1984, 12, 6559