PHYSICOCHEMICAL BASIS OF THE RECOGNITION PROCESSIN NUCLEIC ACID INTERACTIONS, IV. COSTACKING AS THE

CAUSE OF MISPAIRING AND INTERCALATION INNUCLEIC ACID INTERACTIONS*

BY P. M. PITHA, W. M. HUANGt AND P. 0. P. Ts'oDEPARTMENT OF RADIOLOGICAL SCIENCES, THE JOHNS HOPKINS UNIVERSITY,

BALTIMORE, MARYLAND

Communicated by Robert L. Sinsheimer, June 24, 1968

In the first paper of this series,' we reported the interaction between thenucleosides and r(U)n as a model for monomer-polymer interaction. Our re-sults from equilibrium dialysis indicate that only when a threshold concentrationof adenosine was reached did the binding of adenosine to r(U)n to form an orderedstructure take place in a cooperative manner with a stacking energy of 5-6 kcal.Since adenosine associates extensively in aqueous solution to form vertical stacks,2we proposed that these adenosine stacks bind to r(U)n cooperatively through spe-cific hydrogen bonding. Thus, both hydrogen bonding and the stacking forceare essential, with the former more related to specificity and the latter to sta-bility. The interaction of nucleotides and nucleoside triphosphates with r(C)nand r(U)n was reported in the second and third papers of this series.3 4 This samesystem has also been studied by laboratories at the National Institutes of Healthwith different emphasis.5-7

In the present paper, the cause of mispairing and intercalation in nucleic acidinteraction has been investigated.

Materials.-Solutions of r(U). (Miles Laboratories, Elkhart, Indiana) at a concentra-tion of 10 mg/ml were dialyzed against 0.01 M Tris buffer, pH 7.0, for 12 hr in a cold roombefore use. The exact concentration of uridine units in solution was determined fromoptical density measurement at 259 mu by using the molar extinction coefficient of 9.2 X103. Adenosine, guanosine, and cytidine were A grade preparations from Sigma ChemicalCompany, St. Louis, Missouri and were used without further purification. Caffine waspurchased from Eastman Organic Chemicals, Rochester, New York, and was dried at1200 for 24 hr before use. CL-adenosine, HI-caffeine, and HI-BP were obtained fromNuclear-Chicago Corporation, Des Plaines, Illinois, and H'-cytidine and H3-guanosinewere obtained from Schwarz BioResearch, Orangeburg, New York. Before being used,HI-guanosine was checked by paper chromatography for possible contamination ofadenosine.Methods.-Equilibrium dialysis: The experimental procedure of this technique was

described in our previous paper.1 Solutions of r(U). (1.5 X 10-2 M) were dialyzedagainst an invariant concentration of H3-guanosine (1.17 X 10-5 M; 1.1 X 107 cpm/mM),HI-caffeine (1.2 X 10-5 M; 3.52 X 106 cpm/mM), or H3-cytidine (1.13 X 10-5 M;1 X 107 cpm/mM) together with varying amounts of Cl4-adenosine (conc. ranged from1.10-3 M to 2.5 X 10-2 M; 8.7 X 103 cpm/mM) in 0.4 M NaCl, 0.01 M Tris buffer,pH 7.0, at 50 for 10 days. At the end of the equilibrium dialysis period, the distributionof nucleoside inside and outside the dialysis tubing was assayed by a double-countingprocedure in a Nuclear-Chicago ambient temperature liquid-scintillation counter modelno. 722. The concentration of the nucleosides outside the dialysis tubing was consideredto be the concentration of the free nucleosides. The difference in radioactivity betweenthe inside and the outside of the dialysis tubing divided by the total radioactivity (sum-mation of radioactivity from both inside and outside the tubing) gave the percentageof the nucleoside bound.'

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Solubility measurement: This method was used to study the interaction between guano-sine or BP and the r(U)n, taking advantage of their low solubility. Solid H3-guanosine(14 uc/mM after approximate dilution with nonradioactive guanosine) was shaken in0.4 M NaCl, 0.01 M Tris buffer, pH 7.0, for 10 days at 50 in the presence or the absenceof r(U)n (1.5 X 10-2 M). Solubility of guanosine in these solutions was measured fromthe radioactivity of the supernatant after the removal of the excess solid by centrifugationat 104 rpm for 10 min in a Sorval refrigerated centrifuge at 5°. Solid H3-BP (500 mc/mM) was shaken in 0.4 M NaCl, 0.01 M Tris buffer, pH 7.0, for 14 days at 50 in the pres-ence or absence of r(U)n (1.5 X 10-2 M) and adenosine (9.8 X 10-3 M). The excesssolid of HI-BP was removed by filtration through sintered glass filters.8 Radioactivityof the filtrate provides the measurement of solubility. The change in the solubility of acompound from S to S' in the presence of an interacting substance is related to the changein the activity coefficient of this compound from y to y' in accordance with the followingequation: ln(S'/S) = ln(y/y').9 A reasonable assumption is made here that the de-crease in the activity coefficient is due to the formation of a soluble complex of guanosineor BP with r(U)X or with adenosine-r(U). complex. Therefore, the increase in solubility ofthese compounds can be analyzed in terms of solubility increment AS/I, where AS is theincrease of solubility (M) per concentration (M) of the interacting substance I added tothe solution.

Optical rotation versus temperature profile: A Cary 60 recording spectropolarimeter wasused to determine the rotation at 350 m1u of solution housed in a temperature-controlledcell of 1-cm light path.'

Results and Discussion.-(1) Equilibrium dialysis studies: Results of studiesby equilibrium dialysis on the binding of three nucleosides and caffeine to r(U)nat 50 are presented in Figure 1. The original data observed on the per cent ofnucleosides bound to r(U)n versus the input concentration of adenosine are pre-sented in Figure 1A. These data were analyzed further, as shown in Figure 1B,so that the extent of binding is now related to the free adenosine concentration inthe mixture.

A. B. I50 0.5 /

40 0.X0o -°16

M , a, . .I ,, 14

IN30TAR [ ARE AFG.0 - 0.3 12f

liru ilssin00 rs . M N Cl pH70Ua°(A) PeMeofbudaensnA (X-) bon cfeie

20( CA 0.2 8MdI~~~~~GR RGRo,

10 / 0.1 4

/R(O)erusinutcCR CR 2

x10I3 Ix1IO2 IXO3 X1x0-2INPUT AR [m] FREE AR [M]

FIG. 1.-Binding of nucleosides and caffeine to r(U). in equi-librium dialysis in 0.01 M Tris, 0.4 M NaCl, pH 7.0, at 50.

(A) Per cent of bound adenosine, AR (X-X); bound caffeine,CA (A A;bound guanosine, GR (@@;or bound cytidine,CR (O--O) versus input concentration of adenosine.

(B) Amount of adenosine bound per UMP of r(U)X; per centbound caffeine; per cent bound guanosine; or per cent boundcytidine versus concentration of free adenosine. Symbols are thesame as in A,

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(a) Binding of guanosine to the adenosine-r(U)n complex: The adsorption iso-therm of adenosine to r(U)n (Fig. 1B) is essentially the same as that reportedpreviously.' There is no binding of adenosine to r(U)n until a critical concentra-tion of adenosine is reached. Then the binding increases rapidly over a narrowconcentration range until the binding is saturated at the stoichiometry of 2U to1A. This steep transition in the adsorption curve represents a large stackingenergy due to the strong nearest-neighbor interaction. The data in Figure 1were obtained in dialysis mixtures containing an invariant amount of guanosine,caffeine, or cytidine (1.1-1.2 X 10-5 M). The presence of this low concentrationof nucleoside or caffeine (0.1-1% of the adenosine concentration) appears to haveno effect on the adenosine binding curve. The critical concentration of freeadenosine in Figure 1B (10-s M) is lower than that published previously (2 X10-3 M). This appears to be dependent to a certain extent on the preparation ofr(U),f. When the same r(U)n preparation was used, the identical adenosinebinding curve was obtained in the presence or in the absence of guanosine (1 X10-5 M) in the dialysis mixture.'0As shown in Figure 1, no accumulation of guanosine inside the dialysis tubing

is found until the concentration of adenosine has reached a certain threshold.The accumulation begins when about half of the r(U)n sites are saturated withadenosine. The accumulation continues to increase proportionally in response tothe rising concentration of free adenosine even when all the sites of r(U)n havebeen apparently saturated. At the plateau region for the formation of the aden-osine-r(U)n complex (Fig. 1B), the free adenosine concentration is around 7-9 X10-3 M, which is the solubility limit of the free adenosine under identical condi-tions in the presence of excess solid adenosine.' Therefore, for experimentalreasons, free adenosine concentration cannot be increased to a level higher thanthat presented in Figure 1B. The formation of the plateau region after thesteep transition zone in the binding curve together with the stoichiometry fromthe solubility experiments' do indicate that at this level of free adenosine con-centration, the formation of the adenosine-r(U)n complex is very close to themaximum.These results indicate that guanosine does not bind to r(U)" by its own action.

However, guanosine can be bound to the adenosine-r(U)n complex. The mech-anism by which this binding takes place is usually not revealed by this type ofequilibrium study, which provides only the information concerning the initial andthe final stage. However, there are two equilibrium processes being studied herein the same system, i.e., the formation of the adenosine-r(U). complex and theformation of the guanosine-adenosine-r(U)n complex. Comparison of these twoprocesses does provide additional information about the mechanism.To reach the final stage of binding under the equilibrium condition, there are

at least two plausible processes (I and II). In process I, adenosine is first boundto r(U). forming the adenosine-r(U)n complex, and subsequently guanosine isbound to the existing adenosine-r(U)n complex. In process II, guanosine isstacked with adenosine (Al, A2, . .. A.) first, aiid subsequently the guanosine-adenosine stack is bound to r(U),f. These two processes are not mutually ex-clusive and probably both take place to some extent. However, data in Figure

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1B suggest that process I is unlikely to be the dominant one. Since the formationof the adenosine-r(U)n complex is a cooperative process,' the plot of the extent ofthe complex formation (ARb/U) versus free adenosine concentration shows asteep transition between the level of no binding and the level of saturation. Ointhe other hand, the plot of per cent GRb versus free adenosine concentrationshows a line with constant slope, which indicates that once the process becomesdetectable, the extent of guanosine binding to the complex is linearly proportionalto the free adenosine concentration. Therefore, the line of per cent GRb iIlFigure 1B is not parallel to the line of ARb/U, especially near the region of maxi-mal adenosine-r(U)n complex formation. This lack of parallelism indicates thatthe extent of guanosine binding is not directly proportional to the extent of theadenosine-r(U). complex formation. Such a proportionality is required by pro-cess I. This absence of proportionality is especially evident when GRb/ARb isplotted against free adenosine concentration. Instead of a straight line withzero slope, a curve with positive slope is obtained. Therefore, this comparisonfavors process II as the mechanism of binding, i.e., the formation of a guanosine-adenosine-r(U)n complex via intermediate guanosine-adenosine (1,2,...n) stacks.

Previous study in our laboratory has shown that adenosine associates to formstacks in solution with an apparent equilibrium constant of about 4.5 or a freeenergy of about -0.9 kcal at 250.2 This process is exothermic with a AH valueof about -4 kcal."1 Computation based on these values indicates that at 50 theapparent equilibrium constant will be about 7 or the free energy of associationwill be about -1.1 kcal.

Will there be base-pairing between stacked guanosine and uracil of r(U)"through hydrogen bonding? Pairing of guanine to thymine (or uracil) was firstshown to be stereochemically feasible by Donohuel2 and has been recently cal-culated to be rather favorable on theoretical grounds.'3"'5 However, experi-mentally, the pairing of guanine to thymine (uracil) in organic solvents was neverfound in recent studies. Bautz and Bautz'6 and Uhlenbeck, Harrison, andDoty"7 have interpreted their observations to mean that such G-U pairing doesnot exist in oligonucleotide-polynucleotide interactions. In order to evaluatethe relative importance of hydrogen-bonding versus the stacking force upon thebinding of nucleoside in this system we have chosen to study caffeine and cyti-dine.

(b) Binding of caffeine and cytidine to the adenosine-r(U)n complex: Since thereis no hydrogen bond donor site in caffeine, one does not anticipate base-pairingbetween caffeine and uracil. However, the tendency toward self-association ofcaffeine in water is even greater than that of adenosine.9 11 On the other hand,the tendency toward self-association of cytidine is much less than that of aden-osine.2 9 Therefore, though costacking of cytidine with adenosine or purine hasbeen observed,'8 it is expected to be much less than the costacking of caffeinewith adenosine, since the stacking force of the pyrimidine is much less than thatof the purine.9 However, base-pairing between cytosine and uracil, though neverdemonstrated experimentally, is considered theoretically almost as energeticallyfavorable as the A-T pair.'3 14

As shown in Figure 1, there is substantial binding of caffeine to r(U)n in the

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presence of a sufficient concentration of adenosine, but no binding of cytidine isfound under this identical condition. These results clearly show the relativeimportance of the stacking force over the hydrogen bonding in this system.Thus we may conclude that the binding of guanosine is mainly due to its co-stacking with adenosine and not its possible hydrogen bonding with uracil.

In the plot of percentage binding versus free adenosine concentration, the linefor caffeine and the line for guanine both have a constant and similar slope (Fig.1B). For the same extent of binding, for example, 10 per cent, about twice asmuch free adenosine is required for guanosine binding (7.6 X 10-i M) as forcaffeine binding (3.8 X 10- M). In line with the argument presented above(process II), an approximation can be made that this differential requirementreflects the difference between the chemical potential of guanosine-adenosinecostacking and that of caffeine-adenosine costacking in the same environment.The difference in chemical potential is calculated to be about 0.4 kcal in favor ofcaffeine-adenosine costacking by the equation of RT ln (AG)/(ACa) where AGrepresents the free adenosine concentration required for a given degree of bindingof guanosine (for example, 10%) and Aca represents the free adenosine concentra-tion required for the same degree of binding of caffeine. A reasonable approxi-mation is made in this calculation that at this narrow range of dilute concentra-tion (4-8 X 10-3 M), the extent of association of caffeine or guanosine with aden-osine(l,2,....) is linearly proportional to the concentration of free adenosine.On the other hand, if process I is the dominant one, then the equation for thecalculation of the free energy difference between guanosine binding and caffeinebinding to a given concentration of adenosine-r(U)n complex is RT ln [(CAb)/-(CAfree) ]/ [(GRb)/(GRfree) ]. This equation also yields a value of 0.4 kcal. Thisvalue of 0.4 kcal is of some general interest since the free energy of the self-as-sociation of guanosine cannot be measured experimentally because of the lowsolubility of guanosine. The free energy of self-association of caffeine is about- 1.5 kcal at 250. Again, as an approximation derived from an indirect ap-proach, and with an assumption that the values of AH for association do notdiffer greatly among these compounds,1' the self-association of guanosine may bein the neighborhood of -1.1 kcal at 250, whereas that of adenosine was measuredto be about -0.9 kcal.There is another interesting consideration. At the 7 X 10-s M level of free

adenosine concentration in the present system, there is about 1 guanosine boundper 6000 adenosine bound and 1 caffeine per 3800 adenosine. The concentrationof the free guanosine and free caffeine, however, is about 700-fold lower than thatof the free adenosine. If a hypothetical situation is considered in which thecompetition for the site among adenosine, guanosine, and caffeine would takeplace with the same chemical potential, then it appears that adenosine would bepreferred by the r(U)n over guanosine only with a ratio of about 8.5:1, and overcaffeine with a ratio of 5.5:1. The selectivity based on the ability to formbase-pairing with uracil is indeed not very high.

(2) Interaction of r(U). with caffeine and guanosine as studied by optical rotationand solubility measurement: Results in Figure 1 clearly show that there is nointeraction of caffeine or guanosine with r(U)X when these two compounds are

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present alone at low concentration (1.2 X 10-5 M). Can interaction of r(U).with the two compounds be found when they are present in high concentration asin the case of adenosine (Fig. 1)?As shown in Figure 2, the complex of adenosine-r(U). has a very high rotation.

This complex dissociates at higher temperatures with a Tm of about 150, as fullydescribed previously.' In the presence of 1 X 10-2 M caffeine, no such complexwith r(U). was detected. The melting profile of the r(U). in a solution of caffeinewas essentially the same as that found in the absence of caffeine in results pub-lished earlier.' This observation confirms the previous conclusion' that theexistence of a monomer-polymer complex requires both stacking force and hydro-gen bonding through base-pair formation. Our laboratory reported that caffeineis an effective denaturing agent for DNA and for helical r(A)X."9 Subsequently,our laboratory has shown that denatured DNA or the coil form of r(A). has a

0.9

OAR

r. 0.7FIG. 2.-Effect of temperature on the e

optical rotation of r(U). solution (1.5 X n,0 Os10-2 M) measured at 350 mjA: r(U). alone 6 05as control (X-X); in the presence of 1 aX 10-1 M caffeine (@ -); and in the > 0.4 -CONTROLpresence of 9 X 10-2 M adenosine (n

(O--O). 0IAll solutions are in 0.01 M Tris, 0.4 I02 -

M NaCl, pH 7.0.0.1 - CA

0

10 20 30 40TEMP (°C)

much higher affinity for caffeine than the native DNA or helical r(A)".20 Thisdifference of affinity provides the driving force for the caffeine as a denaturant.The present studies further support these previous conclusions and strongly sug-gest that the mode of interaction of caffeine with nucleic acid is by face-to-facestacking with the bases in nucleic acid. In terms of its biological action, caffeineis well known for its mutagenic effect and for its action in causing chromosomebreakage.2'-24 The real mechanism of these biological effects is not well under-stood. If these effects of caffeine are due to its direct interaction with DNA, thenthe mode of the interaction will be a face-to-face interaction of caffeine with thebases by either insertion or intercalation.As for guanosine, it has been reported that in the presence of 7 X 10-2 M of

r(C)., the solubility of guanosine at room temperature was increased to a value ofover 3.5 X 10-2 M from that of about 3 X 10-3 M in the absence of r(C)..'Solubility of guanosine at 5° in 0.4 M salt was determined to be 2.1 X 10-4 M(Table 1). In the presence of r(U)., the solubility is slightly increased to about3.2 X 10-4 M. The value of AS/I indicates that there are only about seven

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TABLE 1. Solubility of guanosine and BP in the presence of interactants.Concentration of

interactant Solubility S/IInteractant (10-2 M) (10-5 M) (10-4)

Guanosine 0.21Guanosine r(U)n 1.5 0.32 73BP 0.01*BP r(U)n 1.5 0.05 2.6BP Adenosine 1.0 0.03 2.0BP r(U)n 1.5 0.13 4.8

Adenosine 1.0

Experiments carried out in 0.4 M NaCl, 0.01 M Tris, pH 7.0, at 50.* This value is higher than those reported earlier8 in our much more extensive investigation (0.02-

0.06 jAM). The qualitative aspect of the conclusion of this experiment is not affected, however.

guanosine molecules solubilized by 103 uracil bases. The mode of interactionbetween the solubilized guanosine and the r(U)" is not clear at present. Thisstudy again shows that very little interaction between guanine and uracil can bedetected, as observed by others.

(3) Interaction ofBP with r(U). and the adenostne-r(U)n complex: On the basisof spectral data and other pertinent evidences, our laboratory recently proposedthat the interaction between BP and the bases in nucleic acid is by the mode offace-to-face stacking.8 The experimental observations listed in Table 1 furthersupport this notion. Solubility of BP is increased in a solution of r(U)n or in asolution of adenosine. At 50 and 0.4 M salt, r(U)n is in a partial helical form(Fig. 2) and there is substantial association of adenosine to form stacks, as dis-cussed above. Thus, BP can be solubilized by interacting with the stackedbases, as concluded in our previous paper.8 When a helical complex is formedbetween r(U). and adenosine, about twice as much BP-can now be solubilized peramount of bases present in the solution when the value of AS/I is considered.As anticipated, the interaction of BP with the adenosine-r (U)n complex is strongerthan that with adenosine or with r(U)n separately.Summary.-Equilibrium dialysis studies indicated that guanosine and caffeine,

but not cytidine, are bound to the adenosine-r(U)n complex by the mechanism ofcostacking with adenosine, whereas optical rotation and solubility studies indi-cated that guanosine and caffeine by themselves do not interact with r(U)n;similarly, the binding of 3,4-benzpyrene to the adenosine-r(U)n complex wasfound to be greater than that to r(U)n. These results show that mispairing andintercalation in nucleic acid interaction are caused by costacking of these com-pounds with the bases in nucleic acid.

We wish to thank Professor Norman Davidson for his very helpful comments on thispaper.

The abbreviations used are: r(U)X, polyuridylic acid; r(C)n, polycytidylic acid; BP, 3,4-benzpyrene; Tris, tris(hydroxymethyl)aminomethane; UMP, uridine monophosphate.

* This work was supported in part by Atomic Energy Commission contract no. AT(30-)-4538.

t Present address: Department of Biochemistry, Albert Einstein College of Medicine,Bronx, New York 10461.

1 Huang, W. M., and P. 0. P. Ts'o, J. Mol. Biol., 16, 523 (1966).

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2 Broom, A. D., M. P. Schweizer, and P. 0. P. Ts'o, J. Am. Chem. Soc., 89, 3612 (1967).3 Ts'o, P. 0. P., and W. M. Huang, Biochemistry, in press.4Ts'o, P. 0. P., and M. P. Schweizer, Biochemistry, in press.5 Howard, F. B., J. Frazier, M. N. Lipsett, and H. T. Miles, Biochem. Biophys. Res. Commun.,

17, 93 (1964).6Howard, F. B., J. Frazier, M. F. Singer, and H. T. Miles, J. Mol. Biol., 16, 415 (1966).7Maxwell, E. S., L. Barnett, F. B. Howard, and H. T. Miles, J. Mol. Biol., 16, 440 (1966).8 Lesko, S. A., A. Smith, P. 0. P. Ts'o, and R. S. Umans, Biochemistry, 7, 434 (1968).9 Ts'o, P. 0. P., I. S. Melvin, and A. C. Olson, J. Am. Chem. Soc., 85, 1289 (1963).0 Huang, W. M., thesis, Johns Hopkins University, Baltimore (1967).11 Gill, S. J., M. Downing, and G. F. Sheats, Biochemistry, 6, 272 (1967).12 Donohue, J., these PROCEEDINGS, 42, 60 (1956).13 Pullman, B., P. Claverie, and J. Caillet, J. Mol. Biol., 22, 373 (1966).14 Nash, H. A., and D. F. Bradley, J. Chem. Phys., 45, 1380 (1966).15 Lunell, S., and G. Sperber, J. Chem. Phys., 46, 2119 (1967).16 Bautz, E. K. F., and F. A. Bautz, these PROCEEDINGS, 52, 1476 (1964).17 Uhlenbeck, O., R. Harrison, and P. Doty, in Molecular Associations in Biology, ed. B.

Pullman (New York: Academic Press, 1968), p. 107.18 Schweizer, M. P., S. I. Chan, and P. 0. P. Ts'o, J. Am. Chem. Soc., 87, 5241 (1965).19 Ts'o, P. 0. P., G. K. Helmkamp, and C. Sander, these PROCEEDINGS, 48, 686 (1962).20 Ts'o, P. 0. P., and P. Lu, these PROCEEDINGS, 51, 17 (1964).21 Novick, A., in Brookhaven Symposia in Biology, No. 8 (1956), p. 201.22 Kubitschek, H. E., and H. E. Bendigkeit, Genetics, 46, 105 (1961).23 Loveless, A., Genetic and Allied Effects of Alkylating Agents (University Park: Penn State

University Press, 1966).24 Kihlman, B. A., Actions of Chemicals on Dividing Cells (New York: Prentice-Hall, Inc.,

1966).

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