THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 21, Issue of November 10, pp. 6719-6731, 1974

Printed in U.S.A.

Netropsin

A SPECIIW PROBE FOR A-T REGIOKS OF DUPLEX DEOXYRIBONWLEIC ACID*

(Received for publication, April 8, 1974)

ROGER M. WARTELL,$ JACQUELYNN E. LARSOS, AND ROBERT D. WELLS

From the Depadment c);f Bioch.em.&ry, Cdlege of Agricultural and Life Sciences, University of Wisconsin, Ma&- son, Wzsconsz-n 53706

SUMMARY

The binding of netropsin to 31 different natural and bio- synthetic DNAs, DNA-RNA hybrids, and RNAs was studied in order to delineate the nucleic acid structural features necessary for binding. The measurements employed were spectral titrations, analytical density gradient centrifugation, thermal denaturation, equilibrium dialysis, and inhibition of in vitro replication and in vitro transcription.

The major conclusions are: (a) in 0.1 M or higher salt con- centrations, netropsin has a marked specificity for DNAs which contain A-T (or I-C) pairs. It binds tightly to two DNAs which contain only A-T pairs and to two DNAs which contain only I-C pairs. However, no measurable binding was found for two DNAs which contain only G-C pairs. (b) Netropsin’s inability to bind to G-C paired regions is a consequence of the Z-amino group of guanine. (c) Netropsin is specific for duplex DNA; no binding was observed to five single-stranded DNAs, three helical RNAs, or two of the three DNA-RNA hybrids studied. (d) Netropsin binds to DNA by a nonintercalating mechanism, since it does not cause unwinding of supercoiled DNA. (e) Netropsin in- hibits in vitro DNA and RNA synthesis by binding to the template or to the primer, or to both. (f) The closest distance between bound netropsin molecules is three base pairs.

A molecular model for netropsin binding in the minor groove of DNA is proposed.

Previous studies (reviewed in Ref. 1) showed that the sequence of nucleotides in a DNA determines the properties of that DNA. This influence was demonstrated by a varietv of physical, chem- ical, and enzymatic measurements on 14 helical biosynthetic DNAs with defined, repeating nucleotide sequences. Our cur- rent studies are aimed at assessing the influence of neighboring structures on each other. Also, we wish to study the transmission of conformational stability (telestability) l

* This work was supported by funds from the National Science Foundation (Grant GB-30528X) and the National Institutes of Health (Grant CM-12275).

$ Postdoctoral Fellow of the National Institutes of Health (GM 50533-02). Present address, Schools of Physics and Biology, Georgia Institute of Technology, Atlanta, Georgia 30332.

A useful tool for these and related studies would be a small molecule which binds to DNA with a specificity complementary to that of actinomycin D. After surveying a number of likely agents, including nogalamycin, phleomycin, quinacrine, distamy- tin, and reticulomycin, we concluded that netropsin could best fulfill this requirement. It binds specifically to helical DNA at A-T pairs but not at G-C pairs. It does not intercalate but may cause a structural distortion. Previous work (Z-5) showed that actinomycin D binds specifically to helical DNA at G-C pairs, causing a structural distortion. The availability of these two probes with complementary specificities permits dual binding studies heretofore not possible. One such study is to evaluate how the binding of one ligand to DNA affects the binding of the other. Studies with natural DNhs and a deoxyribo-oligomer duplex synthesized for this purpose, d(&AJ md(T1&), will be reported subsequently?

Netropsin (Fig. 1) is a highly basic small molecule produced by Streptomyces netropsis. It exhibits a wide spectrum of toxicities, including antibacterial, -fungal, and -viral activities and the in- hibition of DNA or RNA tumor viruses in mammalian cells (for a review see Ref. 6). Zimmer et al. (7, 8) previously concluded from studies on natural DNAs that netropsin bound preferen- tially at ,4-T sites. We have confirmed this observation and have critically evaluated the degree of its specificity with a number of different nucleic acids, Also, the binding parameters of netropsin to DNA were quantitatively determined. Studies on the mechanism of binding to DNA and the mechanism of inhibi- tion of replication and transcription were performed. A model is presented which is consistent with the experimental findings on the netropsin-DNA complex. Similar binding principles may be involved in the interactions between other peptidic substances and DNA (9).

A portion of these studies was described previously (10).

EXPERIMENTAL PROCEDURE

Materials

DNA Polymers-The procedures used for the preparation of the DNA polymers were described. The polymers were the following: (dA-dT),* (dA-dT), (11); (dA),m (dT), (12, 13); (dI-dC),* (dI-dC), (14, 15); (dI),* (dC),, (dG),. (dC), (16); (dG-dC).* (dG-dC), (15, 17); (dT-dG).. (dC-dA). (18); (dI),* (dBrC), (16); (dA- dBrU),m (dA-dBrU). (19); and (dA-dU),m (dA-dU), (20). The homopolymer duplexes (dA),m (dT)., (dG),- (dC),, (dl),. (dC),,

1 J. F. Burd, R. M. Wartell, J. E. Larson, and R. II. Wells, manuscripts in preparation.

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and (dI),* (dBrC). were separated into t,heir constituent single- stranded polymers by alkaline density gradient centrif ugation as described previously (5). The single-stranded polymers were then recombined to give equal molar ratios of the two strands. For (dG ).m (dC ),,, the polymer solut,ion was made alkaline (pH - 12.5) with sodilmz hydroxide and then slowly dialyzed back to neutrality. Only (d(:)n. (dC),, which was prepared in t,his marmel showed a sharp absorption-temperature transition corresponding to that reported previously (16). 1t also had a sharp (.iaussian- shaped peak by analytical density gradient centrifugation in C&O4 solution at 1.500 g per ml.

Single-stranded DNA polymers (dG ),,, (dC),,, (dT)., (dA),, and (dI), were prepared by separating the strands from parent, duplex polymers as described above. The polymers were purified from reaction mixtures by methods previously described (17 ).

Analytical zone-sediment,at,ion st,udies were performed in I .O M NaCl-0.005 M IWTA (pH 7.5) solution (21) on each of the DN.4 duplex polymer preparations. The s:~,~ values ranged from 28.4 for (dI-dC).m (dI-dC). to 4.0 for (dG-dC).. (dG-dC).. The s!o,~ values previously reported (14, 22) are representative of the values for these preparations.

Each of the I>N,4 polymers was analyzed for purity and compo- sition by analytical buoyant density centrifugation, bot,h in the absence and in the presence of added marker DNA. By this method each sample was shown to be free (estimated at less than 3 to 5yQ) of any other c*ont,aminating polymer. The buoyant densities in cesium chloride or cesium sulfate solution, or both, of the DNA4 polymers used in this study are identical with those report,ed previously (14, 2% 24).

(dG),m (dC)ln--The polymer oligomer complex (dG),* (dC)12 was prepared from the DNA homopolymer (dG), and the DN14 oligomer (dC ) 12. (dC)lt (gift of J* F. Burd, Department of Biochemist,ry, University of Wisconsin ), was prepared by prepara- tive gel clectrophoresis of the product of a pancreatic DNase digest of (d@),,.1 It was comprised of at least 800/, (dC)12; the remainder was (dC)ll and (dC)13. Tn order to obtain a duplex, a solution of eyuimolar amounts of (dG), and (dC)lT was made alkaline (pH N_ 1’2.5) and this solution (in a 13-mm diameter vial) was placed under a bell jar at 23” along with another, similar, vial containing 257; HCl solution a After allowing the HCl to diffuse for 24 to 48 hours, the pH of the polymer solution was 7.0 to 8.5. Following this procedure, t)he complex was dialyzed into an appropriate buffer solution (0.007 M NaCl, 5 X 10B4 M EDTA, and 0.001 M phosphate (pIT 7.5)). The (dG>,m (dC)lz complex eluted as one sharp peak in the void volume of a Fephadex G-75 column (80 cm X 1.8 cm) using 1.0 M NaCl solution a,s eluant. A small amount (approximately 10%) of free (dC)12 eluted at V,:Vo = 2.76. Analytical buoyant density centrifugation of (d(G),. (dC)12 in cesium sulfate solution showed a single sharp Gaussian- shaped band (Fig. 5). The ultraviolet absorption spectrum was similar to that of (dG),* (dC)..

The ratio of the two strands in the complex was measured by the absorption spectra of (d(i),* (dC)la in 0.1 N Nash solution. At this high pH, the DNA4 separated into single strands. Con- centrations of the (dG). and (dC)lT strands in moles of nucleotide were evaluated from the equation

Ax = ex(dG )xdG + a(dC)Xdc

where Ah is the absorption at the wavelength A, ,h(dG) and eh(dC) are the extinction coeticients of (dG), and (dC)lz, respectively, in 0.1 N Na0H solution, and x’i is the molar concentration of the ith component. A least squares fit of the previous equation using nine wavelengths between 240 and 280 nm gave a (dG),: (dC)lz ratio of 1.17. The extinction coeficient of (dG),. (dC), (16), 7.4 X lo3 at 253 nm, was used for this complex for derivation of the binding data in Table II and related experiments.

RNA and RNA -DNA Polyrrlers-Single-stranded ribopolymers

(rC)n, (W., (run, Nun were purchased from Miles Laboratories, Inc. After the RNAs were extracted with phenol and dialyzed (17), they were used to prepare (rA),. (rU), and (rI)n* (rC). by combining equimolar ratios of the constituent strands. The DNA-RNA hybrid polymers (rI)nm (dC), and (rA),. (dT), were prepared in a similar manner. (rA-rU),m (rA-rU). was synthe- sized and characterized as described (25).

RNA and RNA-DNA hybrid polymers were analyzed for composition by absorbance-temperature transitions in Buffer A

(0.1 M NaCl-0.001 M phosphate, pH 6.4). All 1’, values were within 2” of literature values (26).

The s;~,~ values for the ribopolymers were: (rU),,, 3.8; (rA)., 1.0;

(rI )n, 8.4; and (IQ., 2.6. These values were obtained by analyti- cal zone sedimentation in 1.0 M NaCl-0.005 M KI)TA solution, pH 7.5. Single-stranded DNA polymers were scdimented (21) in 0.9 M sodium chloride-O.1 M sodium hydroxide solution. Kedimen- tation values for (dI),, (dA)n, (dC),, (d(j),, and (dT), were bet,ween 3.0 and 9 .O S.

The RNA-DNA4 #X174 hybrid (gift of J. I?. Burd) was prepared by transcribing 4X174 DNA (containing amber 3 and ts 411) mutations; gift of B. W. Porter, this laboratory) with the Esche- richia coli I< NA polymerase under reaction conditions similar to those described below. Approximately twice as much RNA was made in the reaction as I>NA which served as template. The product, was purified by repeated extraction with CHCls-octanol and dialysis; 85c;/c recovery was found. The hybrid was purified from free RNA by preparative Cs#Oa den&y gradient centrifuga- tion ; a single peak of hybrid was found at 1.49 g per ml. The final product contained equimolar quantities (+5(s) of the DNA template and RNA product, as judged by spect,ral and radio- act,ivity measurements. Analytical CszS04 density gradient analysis of the purified hybrid showed a single sharp band at 1.489 g per ml.

Naturally Occurring DNAs-E. coli DN*4, Closlridium per- fringens DN,4, and calf t’hymus DNA were obtained from Sigma Chemical Co. and further purified by phenol extraction and dialysis. J&~sct~ccus Euteus DNA was isolated by the procedure of Marmur (‘L7); preparative centrifugatron in a cesium chloride densit,y gradient served as the final st,ep. Crab (dA-dT), DNA was isolated from a crude DNA preparation from Gecarcinus Zaferalis (generous gift of D. Skinner, Oak Ridge National Labora- tory) by cesiurn sulfate density gradient centrifugat,ion in the presence of mercury ion (‘28). Crab (dA-dT), DNA isolated from a crude DNA preparation from Cancer borealis (generous gift of M. Laskowski, Sr., Itoswell Park Memorial Institute) was also employed. Supercoiled Ml3 RF1 DNA was a gift of A. R. Morgan, University of Alberta. All of the above natural DNAs were examined for purity and composition by analytical cesium chlo- ride buoyant density centrifugation. Buoyant density values corresponded to previously established literature values (23) and no detectable arnounts of other nucleic acid, including RNA, were found.

All nucleic acids w-ere normally dialyzed into Hrlffer A. This buffer was used throughout unless specified otherwise.

Ketropsin-The netropsin used in this stlldy was the generous gift of N. Belcher, Pfizer Inc. It was dissolved in Buffer A. The molar extinction coefhcient at 296 nm in Buffer A was 2.02 X lo? This was obtained by comparison with the previously re- ported value (its) in another solvent. The purity of the netropsin was checked in three descending chromatographic systems and by its absorbance spectra (30). Single ultraviolet absorbing spots were found at -IcF = 0.82 in isobutyric acid-concentrated ammonium hydroxide-water (66:1:33, v/v/v); 12~ = 0.05 in 0.1 M

sodium cacodylate-ammonium sulfat,e-1-propanol (600 g of am- monium sulfate in 1 liter of 0.1 M sodium cacodylate plus 20 ml of 1-propanol); and I& = 0.43 in l-butanol-acetic acid-water (4:1:5, v/v/v>, Whatman No. 1 paper was used throughout.

Since dissolved netropsin appeared to undergo a slow degrada- tion, as evidenced by changes in its absorbance spectra, care was taken to check stock solutions periodically. Dissolved netropsin was kept at -20” and was used only over a period of 4 to 6 weeks.

Other netropsin samples were generously provided by F. Arca- mone, Farmitalia; 15. L. Patterson, Lederle Laboratories ; and L. Ninet, Rhane Poulenh. Preliminary tests showed that all samples had properties similar to the netropsin used. Netropsin is probably identical with congocidine (31).

Methods

Absorbance Difference Spectra-The absorbance difference spec- tra of nucleic acid-netropsin complexes were obtained by measur- ing t,he absorbance of nucleic acid plus netropsin, with netropsin as the blank. Polymer solution (1.00 ml of 50 to 80 PM) and 1.00 ml of Buffer A were placed in l-cm light path cuvettes. The cuvettes were weighed and the volume was adjusted if necessary. Equal additions of concentrated netropsin solution (5 X 10m4 M)

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were made to both cuvettes with a lo-p1 Hamilton syringe. A Cary 15 spectrophotometer was employed for all spectral measure- ments; all studies were conducted at 24-26”.

The term AASL/CUNA (used in Tables I and III), at a certain concentration of netropsin/nucleotide, is the observed absorption of DNA at 325 nm plus drug (with the same drug concentration in the blank) divided by the DNA concentration (expressed as molar nucleotide).

All DNA concentrations are in moles of nucleotide (or base pairs if so stated).

Netropsin has no appreciable fluorescence. Spectral ‘I’itrations-Binding isotherms of the netropsin-DNA

interaction were obtained spectrophotometrically with t,he use of lo-cm light path cylindricaicells in a Cary 15 spectrophotometer. A DNA solution. at 8 to 11 !.LM in base pairs, was filtered throllgh a sintered glass funnel to &move dust and pipctted int.o the ceil. The titration was carried out by adding 5- to 10-J aliquots of netropsin (5 x lo-’ M); absorbance at. 325 nm was measured. Since free netropsin also absorbs at 3’25 nm, a similar titrat,ion using the solvent was carried out separately. This solvent titra- tion established t,he backgrormd absorbance of free netropsin at 325 nm.

The concentration of the netropsin added was obtained from the absorption at 296 nm. The concentration of bound netropsirr (06) is given by

Db = AAm/[(t;;15 - t&)10] (1)

where & and ttie are the molar extinction coefficients of free and bound netropsin, respectively, measured at 325 nm, and AA315 is the absorbance difference between the DNA solution and solvent with the same amollnt of total netropsin meastlred in the lo-cm cells. The free net.ropsin concentration, D/, is obtained by subtracting f>b from the total netropsin concent.ration, D,. A $, of 15,500 f 20;. was determined by adding netropsin (t,o 2

PM) to 1-m cells VOntaiIlillg I)NA SOllltiOrlS at 0.25 t0 1.0 mg per

ml. At this high ratio of DNA to nctropsin, it was assumed that all of the drrlg was bound. Calf thymrls I)NA, (dI-dC),.(dI- dC),, and (dA-dT),. (dA-dT), all gave the same $& within the limit stated above.

Care must be taken for these titrations because of the small absorbances measured (B.4 31: 5 0.015 per aliquot). The lo-cm cells were firmly taped into the cell holder. After adding an aliquot of netropsin, the holder was gently rocked, and the ab- sorbance was measured and remeasured 5 to 10 minutes later. I~:qnilibrium was achieved in less than 15 s.

The eq~~ilibrium binding data were plotted as r/D, versus r, where r is the moles of bound netropsin divided by the DNA concentration in base pairs (3’2). From this plot, binding paran- eters were obtained appropriate to a model in which all IIN. binding sites are considered to be independent of each other (33).

The equation from this model is

r/D, = K;,,,(f3,,, - ~1 (2)

where K,,, and H,,, are the apparent binding const.ant and ntlrn- ber of binding sites per base pair, respectively. K,,,, is the nega- tive of the slope, and H,,,, the T/D, = 0 intercept of the linear region of the r/DJ zlerst(s r plot. The limit of the assa; was (U,,,)-l = approximately 75.

Equilibriums Dial?lsis--1‘~yrrilibrium dialysis was carried out by a procedure and an apparatus similar to that described previously (5). Dialysis tubing (No. 8, Union Carbide Corp.) was sand- wiched between two rectangular Plexiglas plates which had holes (s inch diameter X 1 inch) drilled at the juricture of the plates. Since ultraviolet absorbance was used to determine netropsin concentration, only relatively high ratios of free to bound ne- tropsin could be examined. Other details aere as described previously.

AnaLytical Density Gradient Centrifugaiion--Analyt.ical cesium sulfate density gradient centrifugation experiments were per- formed as previously described (24). All runs were performed at 25” at 44,770 rpm. For determining the possible interactions between netropsin and the nucleic acids, experiments were done as follows. A cesium sulfate solution (0.65 ml; initial density 1.470 g per cm3) containing the DNA (G to 8 nmoles) as well as marker DNA was centrifuged to equilibrium. For experiment,s in which the netropsin concentration was varied (Fig. 2), after

the first centrifugation run, additional concentrated netropsin solution (5 to 10 ~1) was added directly to the centrifuge cell by means of a Hamilton syringe, and the DNA buoyant density was again determined.

(d(LdC).. (d(;-dC), and (dA), were used as density markers to measrlre the binding of netropsin to other DNAs. To establish t.hat these t.wo polymers were unaffected by netropsin in cesium sulfate solrltion, the following experiment was performed. The DNA in question was prepared in the appropriate cesiunl sulfate solution and equal volunles Fere added to two cent.rifrlge cells. After centrifuging both cells to equilibrium, ultraviolet photog- raphy established that both DNA bands were in perfect alignment in the two cells. Then 10 to 20 ~1 of buffer were added to one cell and an equal volume of concentrated netropsin was added to the other. After centrifuging the cells to equilibrium a second time, it was found that the 1)NA bands were still in perfect alignment between the two cells. llepeating this procedure to high ne- tropsin concentrations ( -10 PM) indicated that the buoyant der1sit.y of the DNA in question was unaffected by netropsin.

Because of the effects of high salt concentration on I)NA structure (17, 34) and the effect of cesium sulfate on DNA-ligand interactions, these mcasurcments must. be compared cautiously with results in a different. medium. Within these limitations, however, equilibrium htoyant density cent.rifugation provides simple means of assaying for drug binding to small amounts of nucleic acids (5, 35).

Set/i,/le~nlulior~ of Sllpercoiled Dl\rA Glh i\‘efropsin- Sedimenta- t.ion roeffirients were nleasrlred by borlndary sedimentation in a Beckman model I<: rllt raccntrifuge. All rt1ns were performed at 25” with the temperatrire control on “indicate” to avoid thermal convection (3(i). The cell contained a lZmm Kcl-F centerpiece. The rotor speed was 14,770 rpm.

Ml3 1:F 1)NA (0.60 ml of 63 JAM) in Ikffer A was added to the ultracentrifuge cell. The volume was determined by weighing the cell before and after adding the DNA solution. After the sedi- mentation run of the DNA alone, 5 ~1 of netropsin solution (140 P”M) were added to the cell with a Hamilton syringe. The cell was shaken to mix its contents, and the sedimentation determina- tion was repeated. This procedure was continued until 5 ne- tropsin to 1)NA ratios were obt.ained on the same DNA sample. An identical procedrlrc was employed with ethidium bromide and Ml3 l:F 11N.4. Since ethidium bromide was previously shown to change the sedimentation cocfhricnt of closed circular DNA (3G), this provided a control for the netropsin-11X.4 sedimentation experiments.

Abso~Oarccc-‘l’cttl~e~a/Ic~e l’ransitions~llelis roil transitions nere obtained with a (iilford model 2000 spectrophotometer as described previously (17). Thermal transitions of DNA SO~II-

tions with and without netropsin were measured simultaneously. Aliqrlots (10 to 20 ~1) of concentrated netropsin were added to equal volumes of I>K\;A and solvent. Just prior to the melting c,Irve study, heli\un gas was bubbled through the solutions and then they were c*entriftlged to remove dust. I>N4 and solvent solutions, with and nitho\lt netropsin, were treated similarly, and all four solutions were examined simultaneously. DN.4 concentrations of abollt 40 p~ nucleotide were employed. Melt- ing curves nerc normally monit orad at the &,:Lx of the polymer with the use of glass-stoppercd IO-mm light path curettes with a \olumr capacity of 0.6 ml. The solvent was Buffer A.

Ii,\-A l’ol!/~~lernse anrl DSA l’ol!p~erase I/eat/ions-l<NA polym- erase and IIN. polymerasc reactions were monitored by following the incorporation of radioactive ribonucleoside triphosphates or deoxyribonrlcleoside triphosphates into arid-insoluble polynucleo- t.ides as previollsly described (37, 38). The DNA polymerase reaction mixture contained, per ml (total volume, 0.1 ml): 50 pmoles of potassium phosphate (pH 7.5); 5 Fmoles of M&l?; 1 pmole of mercaptoethanol; 1 rmole of each deoxynucleoside triphosphate (Schwartz RioResearch, Inc.) needed to replicate the DNA template; SH-labeled dATP, dGTP, or dCTP (New England Nucliar C&p.); I>NA and netropsin at the roncentra- tions indicated in the fiellres: and 30 units of M. lufe?ls DNA polymerase (38). All ingredients u-ere mixed on ice except for the netropsin. This was added last and the mixture was incu- bated at 37”. Samples (0.010 ml) were taken at the time points indicat,ed on the figures.

The MYA polymerase reaction contained, per ml (total volume, 0.1 ml): 40 rmoles of Tris-HCI (pII 8.6); 4 pmoles of MgClz; 1

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wavelength (mp)

FIG. 1. Spectral determination of DNA-netropsin complex. a, absorption spectrum of calf thymus DNA in Buffer A (-); the difference spectrum was created by adding 0.2 mole of netrop- sin per DNA nucleotide to DNA and blank (- - -). b, the absorp- tion difference spectra of calf thymus DNA-netropsin complexes. AA is the absorption of the DNA-netropsin complex minus the

rmole of MnCL; 12 pmoles of mercaptoethanol; 0.5 &mole each of the appropriate ribonucleoside triphosphate (Schwartz Bio- Research, Inc.); YXabeled rATP, rCTP, or rGTP; DNA and netropsin at the concentrations indicated in the figures; and approximately 66 units of E. coli RNA polymerase (39) (gift of M. Ryan and J. Dodgson, this laboratory). As in the DNA polymerase reactions, the netropsin was added last and the reac- tion was monitored at 37’.

RESULTS

Interaction of Netropsin with DNA-The interaction between netropsin and a variety of synthetic DNA polymers and natural DNAs was investigated by absorption spectra, analytical buoyant density centrifugation, thermal denaturation, and equilibrium dialysis studies. Hence the effect of complex formation on both netropsin and DNA properties was measured. Fig. la shows an absorption difference spectra for netropsin complexed to calf thymus DNA. All of the duplex natural DNAs and DNA polymers examined, except for (dG-dC), u (dG-dC), and (dG). . (dC), which do not bind the drug, gave spectra with similar shapes. The ADS2 for a DNA-netropsin complex was charac- terized by a peak at 325 nm and a general hypochromism in the region of the DNA spectra. Zimmer et al. (7) have reported similar spectra of netropsin bound to natural DNAs. An optical titration in the region from 290 to 340 nm is shown for calf thymus DNA (Fig. lb). An isosbestic point was found at 298 nm. Such an isosbestic point was consistent with, although not proof of, a single netropsin-DNA complex. Isosbestic points near 300 run were also found for Clostridium perfringens and ~liicrococcus Zuteus DNA up to a netropsin to DNA molar ratio of 0.45, the highest concentration studied.

Fig. 2 shows the changes in the analytical cesium sulfate buoyant densities of (dA)..(dT), and (dA-dT),.(dA-dT), as a function of netropsin concentration. This measurement pro- vided a valuable alternate assay for complex formation. How- ever, quantitative interpretation of these data was difficult due to the effects described under “Experimental Procedure.” Simi- lar curves were established for all DNAs listed in Table I; only the saturation value has been listed.

* The abbreviation used is: ADS, absorption difference spectra.

0.06 . ./C --._,

,’ %..

-0.04 -

0 300 320 340

wavelength (rnpl

absorptions of equal concentrations of free DNA and netropsin. The DNA concentration was 70 PM. Netropsin concentrations are given as the molar fraction of DNA nucleotide: - -, 0.12; --, 0.29; -.-, 0.57. Further details are described under “Ex- perimental Procedure.”

30, I

FIG. 2. Effect of netropsin on cesium sulfate buoyant densities of (dA-dT),. (dA-dT), and (dA),. (dT),. The equilibrium buoy- ant densities at zero netropsin concentration were 1.425 g per ml for (dA-dT),. (dA-dT), and 1.421 g per ml for (dA),.(dT),. (dA-dT),. (dA-dT),, O-O; (dA),.(dT)r,, A-A. Other details are described under “Experimental Procedure.”

Table I lists the difference spectra extinction coefficients and buoyant density changes for a variety of natural and synthetic DNAs. Except for (dG-dC),-(dG-dC), and (dG)n-(dC)n, all of the duplex DNA polymers examined showed an absorption difference at 325 nm and a buoyant density change. The bio- synthetic DNAs which contained only A-T pairs or only I-C pairs bound the drug, regardless of the type of nucleotide sequence (alternating purine-pyrimidine or homopolymer duplex). Hence, the presence of A-T pairs was not a requirement for netropsin binding. In addition, substitution of dT with dU or with dBrU, and of dC with dBrC did not markedly change binding proper- ties. The apparent inability of the two G-C-containing poly- mers to bind netropsin in 0.1 M or higher salt concentration must be related to the presence of dG, since dI-containing DNAs bind netropsin. In addition, netropsin binds to (dT-dG), . (dC-dA), AA w, ICmu = 340, under the conditions of Table I. Thus more than one consecutive A-T is not necessary for binding.

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TABLE 1

DNA

Natural DNAsc Cancer borealis (dA-dT),. (dA-

dT),, (37,). Closlridium perfritlgetbs DNA

(30%).

Calf thymus DNA (43ycj: Escherichia coli DNA (507;) Micrococcus luleus DXA (72%).

Biosynthetic polymers (dA-dT),. (dA-dT),

(dA),. (dT),. (dI-dC),. (dI-dC),. : : (dI),. (dC),.

(dGdC),. (dG-dC), (dG),. (dC),

(dA-dU),,. (dA-dU),, (dA-dBrU).. (dA-dBrU), : (dI),. (dBrC),.

rm in 0. ld sodiur

ion

I- G2” 695 23d

76 440 11

81 410 A 86 340 6 94 310 0

61 815 28 66 460 18 54 690 14 43 385 25

112 0 0 101’ 0 0

58 540 26 67 330 33 i3 420 0

~AJ~CDN at 0.2 mol

of netropsin

Per lucleotide

A e

‘2

cszso4 buoyant density

decrease at saturation*

a The absorbance measurements were performed in Buffer A. * All DNAs were saturated at 1 to 5 PM netropsin in these ex-

periments. .411 data were derived from multiple point titrations at different concentrations of netropsin.

c G-C contents are given in parentheses. T,,, values are listed

for the sake of comparison. d Gecarci,lzcs lateralis (dA-dT), was used for this experiment.

e Extrapolated from values at lower salt concentrations.

Bixdi,rg cothstaj/ls for the i,r/eraclzot/ of ~telropnirl wi/h DNAs

Studies wcrc carried out in Buffer A; all data were derived from spectral titrations (see “I~~aperimentnl Procedure”). (U,,,,)-* =

apparent number of base pairs per binding site. K,,,, = apparent binding constant. The standard deviation on the stated values is &lO’i;, for (!1,,,,)-l nnd is f20% for B,,, K;,,,,.

Thermal denaturation studies were performed on several of the DNAs in Table I. The T, of (dh-dT) n. (d-1-dT) n increased 23” by adding 0.2 mole of netropsin per mole of nucleotide (Fig. 4). Increases of approximately 23” also were found for (dI),. (dBrC) n and (dI-dC),.(dI-dC),. Because the melting tempera- tures of the G-C DNA polymers are greater than 100” under these conditions, it was not possible to study the effect of netrop- sin on their denaturation.

Natural DNAs Clostridium pcrfringetis DNA.. 8.6 Calf thymus DN.4 11.0 Escherichia coli I)NA. 14.4 Micrococcus l&us DNA. 34.5

Biosynthetic DNAs

(dA-dT),. (dA-dT),. 4.8 (dA),. (dT),. 4.9

(dI),. (dC),. The ADS and buoyant densities of five single-stranded DNXs

((dh)., (dC)., (dT)., (dG),, and (dI),,) were examined with varying amounts of netropsin. None of the single-stranded polymers showed buoyant density changes, even at concentra- tions of netropsin saturating for binding DNAs (5 PM). Except for (dG)., no change in the ADS was observed for the ‘single- stranded DNAs up to a molar ratio of 0.25 netropsin per DNA nucleotide. For (dG)., hypochromism did occur in the 230 to 315 nm region. No absorbance change was observed between 320 and 340 nm. This spectral difference with (dG), may be due to some weak interaction (to be described later).

(dI-dC),. (dI-dC),. 1. : 5.3 4.6

Halogenated biosynthetic DNAs

(dI),. (dBrC), 4.7 (dA-dBrU),, (dA-dBrU),. 5.3

Test for determinant of binding specificity (dG),. (dC)lz. 33.0

2.65 X lo6 0.39 x 106

1.3 x 106 0.09 x 106

4.0 x 106 4.9 x 105

2.3 X 106 4.8 X lo6

4.9 x 106 2.3 x 106

3.5 x 10”

An ADS similar to that found with (dG),, was observed for a preparation of equimolar strands of (dG), and (dC), mised together with no annealing step. .iuthentic duplex (dG),. (dC)., prepared as described under “Esperimental Procedure,” showed no change in its ADS. The absorption studies shown in Table I were done in 0.1 M NaCl solution. At lower salt concen- trations (0.01 M Na+), a small amount of netropsin binding to (dG-dC),.(dG-dC), was indicated by ADS changes (shown be- low). These changes were considerably less, however, than the changes observed for the A-T Dolvmers in the same solvent.

Binding Isotherms-Quantitative equilibrium binding measure- ments were performed on a variety of DNhs in order to under- stand better the binding specificity. A typical Scatchard plot is shown in Fig. 3. Although the independent site model (Equa- tion 2 under “Methods”) is not appropriate for most DNh-ligand interactions, we employ the model’s binding parameters to char-

acterize the Scatchard plots. They are obtained readily and permit a relative comparison of netropsin binding to different DNAs.

The intercept of the linear region of the binding curve with the horizontal axis gave B,,,, the apparent number of binding sites per base pair. The intercept with the vertical axis yielded

hJLw, Km defined as the apparent binding constant.

I I Table II lists values of (B,,,)-' and KappBapD evaluated for 11

3s

2.c

8 x

6 ‘r I.(

I 1 I 1 02 04 06 08 .I0 12 .I

r

6723

14

FIG. 3. Scatchard plot for the binding of netropsin to Clos- Iridiunb perfringens DNA. The three symbols (A, 0, l ) cor- respond to three separate experiments. r is the moles of bound netropsin per base pair concentration and DJ is the concentration of free netropsin. Further details are given under “Kxperimental Procedure.”

TABLE II

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DNAs from Scatchard plots. This designation of the apparent binding parameters has been used to facilitate a later comparison with a theoretical model of ligand-DNA interaction (33). For the four natural DNAs, (&J1 increases with decreasing A-T content. This result agrees with previous studies showing netropsin’s preference for A-T-rich DNAs (7, 8).

The four synthetic DNA polymers (dA-dT),.(dA-dT),, (dA),.(dT),, (dI-dC)..(dI-dC),, and (dI),.(dC), all showed similar binding parameters (Table II). This result demonstrated that netropsin can bind with equal affinity to I-C and to ,4-T base paired sequences. The inability of netropsin to bind (dG),.(dC), or (dG-dC) ,, . (dG-dC), (Table I) was apparently due to the 2-amino of guanine in the narrow groove of the DNA duplex. This observation was reinforced by the result that (dI), (dBrC) ,, and (d-4-dBrU),. (dA-dBrU), bound netropsin with an affinity similar to the A-T DNA polymers (Table II). Placing a bromine on position 5 of a pyrimidine (located in the deep groove of the DNA helix) had little or no effect on netropsin binding.

Equilibrium dialysis studies also were used to examine the binding of netropsin to calf thymus DNA, M. luteus DNX, (dG-dC),.(dG-dC),, and (dG),. Because of the difficulty in measuring small quantities of free netropsin, equilibrium dialysis could not be used in the important linear range (T --f 0) of the Scatchard plot. However, binding data from optical titrations and dialysis studies did coincide in the r = Bnpp region of the Scatchard plot for both calf thymus DNA and M. luteus DNA. This result supports the validity of the optical titration method. Dialysis experiments with netropsin and (dG-dC) n. (dG-dC) n also added confidence in the optical titration method. Dialyzing

Binding determinations showed that (dG), . (dG) 12 bound only a small amount of netropsin. Optical titrations yielded the ap- parent binding parameters shown in Table II. These data are near the limit of the spectral assay employed. However, these and ADS data consistently showed a slight interaction between netropsin and (dG) n. (dC) 12. Thermal denaturation studies buttressed this contention. Fig. 4 shows the melting behavior of (dG).. (dC)12 in the presence and absence of netropsin. The T, is increased approximately 1.5“ when 0.2 mole of netropsin

1.533 1.448

’ 1

‘rn

1.534

1 1.448

1

:

Density (g/ml) 50 60 70 80

the same concentrations of netropsin with (dG-dC),. (dG-dC), and (dA-dT),. (dA-dT), showed no netropsin binding to (dG- dC) ,, . (dG-dC) “, whereas netropsin binding to (dA-dT) ,, . (dA- dT), was clearly evident. Equilibrium dialysis of netropsin with (dG), exhibited little, if any, binding (D, N Dt/2) down to a value of T = 0.0025 mole of netropsin per mole of nucleotide.

Test jor Determinant of Binding Specificity-The studies de- scribed above show that netropsin has an apparent specificity for binding at A-T-rich sites, since it binds to all DN.4s except those containing only G-C pairs. However, dI- and dC (or dBrC)- containing polymers bind as well as A-T-containing DNAs. The two common features of the -4-T and I-C polymers are (a) the absence of the “third” hydrogen bond found in the minor groove of the G-C DNAs and (b) their low thermostabilities compared with the G-C DNAs. To distinguish between these two possi- bilities, netropsin binding to (dG). (dC)12 was examined. Because of the shortness of the dC strands, the T, of this duplex was 65.8 + 0.5” in Buffer h (Fig. 4), approximately 36” lower than the T, of (dG),, . (dC),. Hence, this polymer oligomer has a T, corresponding to that of DXAs which bind netropsin (Ta- bles I and II) but it still possesses three hydrogen bonds.

TEMPERATURE (“C)

FIG. 4 (leff). I’ffect of netropsin on absorbance-temperat\Ire transitions of (dLdT),. (d.\-dT), and (d(i),,. (dC),%. a, helix coil transition curves of (di-dT),. (d.4-dT), in Buffer A without netropsin (O--O) and with 0.2 mole of netropsin per DNA nucleotide (O---O ). The absorbance was monitored at 260 nm and the relative increases in absorption, A~,,,l/Ai,i~,,,l, were 1.45 with netropsin and 1.40 without netropsin. b, helix coil transi- tion cllrves of (d(i),. (dC),? in Buffer A without netropsin (O--O) and with 0.2 mole of netropsin per DN.4 nncleotide (O--O). Absorbance was monitored at 275 nm, and the

relative increase in absorption for both cases was approximately 1.3. Other details are given under “I~:xperimental Procedure.”

FIG. 5 (rig&). Density gradient strldics on the ability of (d(;),.(dC)12 to bind netropsin. a, Illicroderlsitometer tracing of an ultraviolet photograph of the resium sulfate equilibrium buoyant density run with (d(i),,. (dC)Ir (p = 1.533 g per ml (cwss- ha~cherl). (d(;-dC),,.(d(;-dC), served as a marker (p = 1.448 g per ml). b, same as in a except that 5 PM netropsin was added to the cell prior to the run. See “ISxperimental Procedure” for further details.

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TABLE III Studies on the binding of netropsin to RNAs and

RNA-DNA hybrids

RNA

(rA).. (rU),. (rA-rU),. (rA-rU), (rA)n. (dT), . (rI)n- (rC),. . @I),. (dC). +X174 RNA-DNA hybrid

D NP, not performed.

I/ -

AAJ~CRNA at 0.2 mole of netropsin per

nucleotide

IT , at 0.2 mole of netropsin per

nucleotide

0 <0.5”

0 <0.5 340 +1.5

0 <0.5 0 <0.5 0 NP,=

per nucleotide was added. This may be compared (Fig. 4) with the 23” increase in T,,, for (dA-dT),.(dA-dT),, a DNA which binds netropsin well.

Fig. 5 shows the buoyant density profile of (dG).. (dC),z in cesium sulfate with and without netropsin. No change in the buoyant density value of 1.533 & 0.001 g per ml was observed at netropsin concentrations saturating for other DNAs. Since this DNA binds only a small amount of netropsin and since high salt concentrations decrease KapDBapp (see below), this finding is consistent with the other measurements. Parenthetically, this is the first reported case, to the best of our knowledge, of a poly- mer-oligomer complex which banded in a density gradient.

These determinations indicated that a small amount of netrop- sin binds to (dG),.(dC)12. However, this amount was consid- erably less than the amount bound by A-T and I-C DNA poly- mers. Z,n vitro replication and transcription studies with this DNA as template agree with this conclusion (data presented below). The ability of netropsin to bind to A-T and I-C cannot be determined solely by their relatively low thermostabilities, since (dG), . (dC)i* has a T, comparable to that of A-T and I-C DNAs. Hence, the presence of the 2-amino group of guanine (or the “third” hydrogen bond) in G-C-containing DNAs ap- parently provides a hindrance for netropsin binding.

Studies on Interaction of Netropsin with RNAs and RNA . DNA HyKds-Table III shows the results of attempts to bind netrop- sin to several synthetic RNA and RNA. DNA hybrid polymers. In 0.1 M sodium ion no indication of netropsin binding was found for (rA),.(rU),, (rA-rU),.(rA-rU),, (rI)n.(rC)n, and (rI)n. (dC),. With up to 0.2 mole of netropsin per RNA nucleotide there was no change in the ADS or denaturation curves of these polymers. These results agree with the general conclusions of Zimmer et al. (8) for RNA nonbinding in 0.1 M salt. .

A somewhat unexpected result was found for (rA),-(dT),. The ADS of this polymer showed the typical characteristics for netropsin binding to duplex DNA. Formation of a weak netrop- sin-(rA),. (dT)n complex also was indicated by an increase of 1.5’ in the midpoint of the melting curve for (rA),. (dT),, with netropsin. An optical titration of netropsin with (rA)n. (dT), was carried out to estimate the binding constants. the &

By using found for DNAs, a Scatchard plot yielded values of

RwpKapp = 2.2 x lo4 MS’ and (B.&l = 20. A hybrid of single-stranded 4X174 DNA and its RNA transcript also showed no affinity for netropsin by ADS. Although (rA)n. (dT). se- quences in this DNA might be expected to bind netropsin, it is not known whether long dT sequences exist in 4X174 DNA or how long they must be to assume the structure of the (rA)n- (dT), polymer.

It is interesting to note that the duplex RNA polymers which

2601

16~o~~~ 002 004 0.06 008 0.10

MOLES DRUG/MOLES DNA

FIG. 6. Eflect of netropsin and ethidium bromide on the sedimentation coefficient of Ml3 RF1 DNA. Procedures for the preparation of DNA-drug mixtures and details of the experiment are given under “Experimental Procedure.” O-O, netrop- sin; l -- l , ethidium bromide.

do not bind netropsin have relatively low T, values. Thus the inability of netropsin to bind these RNA duplexes is due to the lack of some structural requirement (possessed by DNA but not RNA) rather than insufficient thermolability or “looseness” of the polymers.

In addition, the single-stranded RNAs (rA),, and (rU), showed no interaction with netropsin by the ADS, under the conditions of Table I (data not shown). Analytical buoyant density analyses could not be employed for monitoring interactions with RNAs because of the precipitation problems encountered in these types of studies (25).

Mechanim of Binding-The results described above have pro- vided information on the nucleic acid structural features neces- sary for the binding of netropsin. To probe further into the mechanism of binding to DNA, the interaction of netropsin with supercoiled Ml3 RF1 DNA was studied. Molecules which in- tercalate into DNA can unwind and then rewind right-handed supercoiled DNAs as a function of ligand concentration (36,40). This effect can be monitored by measuring the fall and rise in the sedimentation velocity of a supercoiled DNA as the amount of bound ligand is increased. A molecule which binds to DNA but does not unwind the double helix will not show this behavior. Its sedimentation is essentially unchanged.

Fig. 6 shows the inability of netropsin to influence the sedi- mentation behavior of Ml3 RF1 DNA. No significant change was found in the sao+, of the Ml3 DNA over a concentration range from 0 to 0.56 mole of netropsin per mole of DNA nucleo- tide. A control experiment with ethidium bromide showed the typical fall and rise in the s 20,~ value of the Ml3 DNA; thus, the DNA was in the supercoiled form. Netropsin did bind to super- coiled Ml3 DNA, as indicated by ADS (at 0.1 mole of netropsin per DNA nucleotide, AAs Co,, = 350). These results imply that netropsin does not intercalate or unwind DNA upon binding. A similar conclusion has been reached by M. J. Warings with supercoiled PM2 DNA.

3 M. J. Waring, personal communication.

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t-50,

C NETROPSIN/CDNA

FIG. 7. Effect of salt concentration on (dG-dC),+(dG-dC),- netropsin complex. Absorbance at 330 nm was measured as a function of netropsin concentration at the indicated salt con- centrations. The- solutions were prepared by exhaustively dialyzing the DNA versus the indicated salt solution (each con- taining 1 X 10e6 M EDTA (all at pH 6.5)). Doubly distilled and de-ionized water was used throughout and other previously described mecautions (17) were emnloved. IdG-dCL.a (dG-dCL . I - , I -

concentration was 40 to 50 PM nucleotide for all experiments. AA corresponds to the absorbance of (dG-dC),+ (dG-dC), and netropsin minus the absorbance of an equal concentration of netropsin. Other details of the absorption difference procedure are given under “Experimental Procedure.”

It was mentioned earlier that decreasing the sodium ion con- centration of the solvent enables netropsin to bind the G-C polymers. Fig. 7 shows the absorption change in the (dG-dC).. (dG-dC),-netropsin complex at several salt concentrations. Solvents with 0.01 M sodium ion or less show an absorption differ- ence spectrum resembling that in Fig. la. As the sodium ion concentration decreases, the plateau of the absorption change around 325 nm increases. These results show that electrostatic interactions are an important part of netropsin-DNA interaction, as expected from the structure of netropsin. Examination of netropsin-(dh-dT),.(dA-dT). interaction shows a similar in- crease in the plateau of absorption change as sodium ion concen- tration is decreased. However, the LIA&C&A was 3 to 4 times as great as that found for (dG-dC). . (dG-dC), under the same conditions.

Pohl and Jovin (34) indicated that (dG-dC).. (dG-dC), has a different conformation at very high salt concentrations than at low concentrations. To determine whether this alternate struc- ture would bind netropsin, ADS was performed in 2.0 M NaCl solution; no binding was observed.

Conformation of Netropsin-DNA Complex-On the basis of large changes in the circular dichroism of DNA-netropsin com- plexes, Zimmer et al. (41) have suggested that netropsin causes large changes in DNA conformation. Reinert (42) has inter- preted viscosity studies on the netropsin-DNA complex to indi- cate substantial changes in DNA conformation upon the binding of the first few netropsin molecules. Recent studies by wide angle x-ray scattering from gel-like solutions of calf thymus DNA with netropsin’ indicated little or no change in DNA conforma- tion.

To determine whether conformational changes were suggested by the netropsin-DNA binding curves, a comparison was made

4 D. Carlson and W. W. Beeman, unpublished data.

TABLE IV

Prediction of natural DNA (B,,,)-’ values from various models of

netropsin binding sites The four models examined were: Model 1, one A-T pair; Model

2, two A-T pairs; Model 3, three A-T pairs; Model 4, three A-T pairs for one site and two adjacent A-T pairs and a G-C pair for the second site. Results were calculated from equations under “Appendix”: Model 1, Equation A-6a; Model 2, Equation A-Sa; Model 3, Equation A-1Oa; Model 4, Equation A-12a. 2 = 3 for all models, p = 6.0 for Model 4.

Predicted fBapp)-l

DNA Experimental

Model Model 2 ’

Model Model m%pp

1 3 4 -__-___

Closlridium perfrin- gens.. . . . 5.5 6.8 9.8 8.4 8.6

Calf thymus. 5.7 8.1 13.5 10.6 11.0 Escherichia coli. . 6.0 10.1 20.0 14.0 14.4 Micrococcus luteus. . 7.3 21.8 78.0 34.8 34.5

between the binding data from Table II and an allosteric model of DNA-ligand interaction (33, 43). In this model, all of the base pairs of a homogeneous base pair DNA polymer are equiva- lent potential binding sites. Each netropsin is either free in solution or bound to the DNA. When bound, each netropsin has a self-exclusion length, 1, and binds with an equilibrium con- stant K. The length I is the closest possible distance, measured in base pairs, between 2 netropsin molecules bound to DNA. The self-exclusion length can be interpreted as a combination of two effects; the number of base pairs which are physically covered by a netropsin molecule, and the region of DNA around a bound netropsin which is sufficiently distorted so that it is not recog- nizable as a binding site. Although it is not possible to separate these components, if 1 is found to be larger than the longest di- mension of netropsin, it would be evidence for conformational distortion of DNA.

For a homogenous DNA polymer, Zasedatelev et al. (43) have shown that for 1 much smaller than the length of the polymer

(Ba&-l = 21 - 1 Pa)

K = &ppB.pp (3b)

From Equation 3 and Table II it is apparent that a value of 1 = 3 base pairs gives the best fit for all of the DNA polymers examined. Since molecular models of netropsin show that it may span approximately three base pairs, 1 = 3 is consistent with netropsin not causing a pronounced conformational distortion.

To compare the above model of DNA ligand interaction with the natural DNA data, we assume a random sequence of A-T and G-C pairs. Gurskii et al. (44) have formulated relationships between KappBapp and (Bar&l and the parameters of the allosteric model for a random sequence DNA. Under ‘(Appendix” we have used their formulation to derive equations for KsppBapp

and ‘(B,,)-1 in terms of the binding parameters employing various assumptions regarding netropsin’s binding site or sites. We first assumed that netropsin binds to only one type of bind- ing site. Table IV lists the predicted (B.&-r values assuming one, two, and three consecutive A-T pairs per netropsin binding site. The values were obtained by using Equations A-6a, A-8a, and A-lOa, assuming .I = 3. The experimental results were not correctly predicted by any of these cases. The predictions for two consecutive A-T pairs per netropsin site gave the closest fit to the data. Employing the actual nearest neighbor fre- quency data (45) for M. luteus DNA in Equation A-8a increased

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0 t5 30 45 60 90 120

(dG - dC)n (dG - dCh template

0 15 30 45 60 90 I20 Time (minutes)

the predicted (B,,,)-1 to 24.5. This, however, is still outside the experimental error limits.

A simple explanation for the lack of fit in the above cases is that net,ropsin binds to different ,4-T sites with different intrinsic binding constants. Although netropsin’s ability to bind to (dT- dG) n m (dC-d A) n implied the necessity of only one isolated A-T pair, neighboring base pairs may influence the binding constant. This can be demonstrated by assuming two types of sites: A-A-A, three consecutive A-T pairs, and B-A-A or A-A-B, two consecu- tive A-T pairs with an adjacent G-C pair. The A-,4-A site has a binding constant 13 times the binding constant K of the

A-A-B or B-A-A site, Equations A-12a and A-12b give the predicted (&pp)-1 and KappBapp values. Table IV shows the predicted (&,p)-l values for 2 = 3 and 13 = 6. It should be emphasized that the above combination of sites and binding constants is not unique. However, the agreement indicates that strong netropsin binding sites involve two and three A-T pairs. By employing the two-site model (Equation A-12b) with p = 6, a comparison was made between the predicted and experimental ratios of KnppBapp (not shown) + The disagreement was substantial. The reason for this discrepancy is not known. Possible causes are the inadequacy of the model and/or systematic errors in thz KappBapp values. In particular, the &ppBapp value of calf thymus DNA does not follow the trend of the other DNAs.

Inhibition of RNA and DNA Synthesis in Vitro-Zimmer et al. (7) previously showed that netropsin inhibited the in vitro synthesis of DNA by DNA polymerase from Ehrlich ascites cells and the synthesis of RNA by E. coli RNA polymerase. Both of these reactions employed calf thymus DNA as templates. Thus, it was not possible to determine whether the inhibition was due to netropsin interacting with the template or the en- zymes. The reactions shown in Figs. 8 and 9 were allowed to occur to elucidate this point.

Fig. 8 shows the results from in vitro DNA synthesis reactions employing the :\I. Luteus DNA polymerase with (dil-dT),,. (dA- dT) n and (dG-dC),*(dG-dC), as templates. At both 0.2 and 2.0 moles of netropsin per DNA nucleotide, the incorporation of deoxyadenosine triphosphate into (d A-dT) n . (dA-dT) n polymer was completely inhibited. With (dG-dC), m (dG-dC), as tem- plate, no difference (within esperimental error) was observed between the synthetic rate with zero netropsin and with 0.2 mole of netropshl per DNA nucleotide. Even at a molar ratio of 2.0 netropsin to DNA nucleotide, no significant charlge OC-

curred in the rate of DNA synthesis with the (dG-dC), l (dG-dC), template. These experiments conclusively showed that the inhibition of DNA synthesis by netropsin was due to its binding to DNA and not to the polymerase.

Time (minutes)

FIG. 8. Effect of netropsin on the kinetics of incorporation of nucleo- tides into DNA by Micrococcus luleus DNA polymerase. (dA-dT), v (dA-dT), (76 nmoles per ml) and 59 nmoles per ml of (dG-dC)./(dG-dC). in nucleotides were the template levels used, Netropsin concentra- tions are given as the molar fraction of DNA template: e--e, 0; A--A, 0.2; and O--o, 2.0. Other details are described under “Experimental Procedure.”

= 300 - (dA-dT)n (dA-dTh template . E \

z q

0 I5 30 45 60 Time (minutes)

0 I5 30 45 60 Time (minutes)

FIG;. 9. Wect of netropsin on the kinetics of incorporation of ribonucleotides into 1<NA by the Escherichia coli 1C,NA polym- erase. (dii-dT).g (dA-dT), (76 nmoles per ml) and 56 nmoles per ml of (dG-dC),* (d(f-dC). in nucleotides were the template levels used in the two plots. Netropsin concentrations are given as the molar fraction of DNA nucleotide: @---a, 0; &--A, 0.2; o-0, 2.0. Other details are described under “Experimental Procedure. ”

Fig. 9 shows a similar studv which employed these two DNA polymers as templates for i. coLi RNA polymerase reactions. The inhibition of the rate of RNL4 synthesis with a (dA-dT),-

l (dA-dT), template, at 2.0 moles of netropsin per DNA nucleo- tide, was reduced at least lo-fold. RNA synthesis with (dG- dC) n. (dG-dC) n was reduced by 15 to 20 y0 at most under the same conditions. This small amount of inhibition of RNA synthesis from the (dG-dC), . (dG-dC), template may be genuine and due to a small amount of netropsin binding in the low ionic st,rength Tris-HCl buffer.

Thus, the inhibition of RNA synthesis from DNA is due to its effect on the DNA template rather than the enzyme, as concluded for the DNA polymerase reactions.

A study of the polymerization reactions using (dG), l (dC) 12 as a template-primer was performed also. Results from the physio- chemical studies (see above) indicated that netropsin bound only weakly, if at all, to (dC&. (dC)lz. Polymerization reac- tions with and without netropsin were undertaken to add a different type of evidence supporting this conclusion. Fig. 10 shows the RNA polymerase and DNA polymerase reactions for (dG), l (dC)l? at different levels of netropsin. Fig. 10, A and B depicts the in vitro incorporation of deoxyribonucleotides using M. luteus DNA polymerase. The results were independent of which triphosphate was monitored; only a small amount of inhibition was noted at 2.0 moles of netropsin per DNA nucleo- tide. Synthetic reactions with E. coli RNA polymerase and

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TIME (MINUTES)

FIG. 10. Effect of netropsin on the rates of DNA and RNA synthesis using (dG),. (dC)lz as a template. A and B, kinetics of incorporation of deoxyribonucleotides into DNA by Micrococcus luleus DNA polymerase and dG,.dClz at a template level of 67 nmoles per ml in nucleotides. Netropsin concentrations are given as the molar fraction of DNA template: l -- l , 0; A--A, 0.2; C----O, 2.0. Other details are described under “lcxperi- mental Procedure.” C and 13, kinetics of incorporation of ribo- nucleotides into RNA by E. coli RNA polymerase. Template and netropsin concentrations are as stated for A and U. Other details are described under “Experimental Procedure.”

ribonucleoside triphosphates are shown in Fig. 10, C and D. Lit- tle or no inhibition of the reactions occurred at 0.2 and 2.0 moles of netropsin per DNA nucleotide. Hence, these composite results show that netropsin has little or no effect on the polym- erization reactions ternplated by (dG), (dC) i2 and are consistent with the physical studies.

The ability of netropsin to inhibit select,ively t.he degradation of DNAs with defined sequences by exonuclease I* is in agree- ment with the results presented herein.

DISCUSSION

Structural Features Required for Netropsin-DNA Interaction

At least three determinants govern netropsin binding to DNA: (a) an electrostatic interaction, probably between the guanidino and propionamidino groups of netropsin and the phosphate groups of DNA; (b) a steric interaction in the minor groove of DNA influenced by the 2-amino group of guanine; and (c) con- formational features particular to a DNA duplex. Each is described separately.

Electrostatic Interaction-An examination of the structure of netropsin reveals that electrostatic interactions would be ex- pected in netropsin-DNA binding. The highly positive end groups of netropsin provide sites for interactions with the nega- tively charged backbone of DNA. Fig. 7 demonstrated that lowering the ionic strength of the solution enabled netropsin to bind (dG-dC),.(dG-dC),, a DNA which showed no binding at higher salt concentrations. The binding for (dA-dT), . (dA-dT), and (dI-dC). . (dI-dC), also increased with decreasing ionic strength. In addition, previous work with natural DNAs and RNAs showed an increase in netropsin binding with decreasing ionic strength (8). Evidence for the involvement of the highly charged end groups of netropsin was provided by studies with netropsin analogues (46).

Although electrostatic forces between the DNA phosphates

and netropsin are involved in netropsin binding, these forces cannot be the major determinants of netropsin’s specificity. In 0.1 M NaCl solution, netropsin does not bind (dG-dC),, .(dG-dC), and (dG)*.(dC)“, whereas it strongly binds (dA-dT),.(dA-dT), as well as other DNA polymers. Since the ionic environment of the phosphates of all of the DNAs is similar, other factors must govern netropsin’s A-T specificity.

Influence of Guanine-The presence of the 2-amino group of guanine directly or indirectly inhibited the formation of a netrop- sin-DNA complex. Netropsin bound to (dI-dC)..(dI-dC), and (dI),a (dC), to the same extent as the A-T DNA polymers. The only feature missing on both A-T and I-C pairs, which is present on G-C pairs, is the a-amino group of the purine and its hydrogen bond with the carbonyl oxygen of cytosine.

Two possible explanations for the influence of guanine were considered. First, the 2-amino group of guanine could interfere with netropsin binding by steric hindrance. This would impli- cate the minor groove of DNA as a site of netropsin-DNA inter- action. An alternate way to account for the inhibitory effect of guanine’s 2-amino group is the increased thermostability of G-C DNAs. The third hydrogen bond inhibits netropsin bind- ing by decreasing structural fluctuations of the DNA chains. Netropsin can bind A-T and I-C DNA polymers because of their lower thermostability. Unlike the previous consideration, this hypothesis does not localize the netropsin-DNA interaction.

To test whether thermostability or the minor groove region of DNA was involved in netropsin binding, studies were per- formed on (dG), .(dC)iz. This polymer-oligomer duplex had a T, in 0.1 M NaCl solution similar to that of binding DNAs, but it still possessed the hydrogen bond in the minor groove. Since only a small amount of netropsin binding to (dG), . (dC)iz was observed by several criteria, it. was concluded that the presence of the 2-amino group on G (in the minor groove of a DNA duplex) tends to disallow netropsin binding. This could be due to steric hindrance of the 2-amino group or the interference caused by its hydrogen bond to the carbonyl oxygen of cytosine. Such interference might disallow this oxygen to form a specific bond with netropsin. This oxygen is available for hydrogen bonding in both A-T and I-C base pairs.

The fact that (dA-dBrU),.(dA-dBrU). and (dI),.(dBrC), bind netropsin is consistent with the notion of netropsin’s inter- acting in the minor groove of DNA. The bromine atoms, with large van der Waal radii, occupy the major groove.

Znjluence of Nucleic Acid Conformation-A third factor which is probabIy involved in netropsin’s binding specificity is the nucleic acid conformation. Evidence for this is the inability of netropsin to bind (rA-rU),. (rA-rU), and (rA),.(rU),, whereas the DNA polymers with identical sequences do bind. The most probable explanation for this behavior is the conformational differences between the RNA and DNA helices. X-ray diffrac- tion analyses (47, 48) have shown that the RNA helices are different from those of the DN.4 polymers.

Appreciable differences in chemical and physical properties between the DNA polymers have been documented (for a review see Ref. 1). However, it is apparent that these differences do not influence netropsin binding, since even (dI-dC),.(dI-dC),, which has the most unusual properties (15, 49), binds normally.

Model of Netropsin-DNA Complex

The following features of the complex were considered when building a model for the netropsin-DNA complex. (a) Only one major binding mode exists. Studies with the natural DNAs and the periodic duplex DNAs indicated a single netropsin-DNA

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FIG. 11. Preliminary model of netropsin-DNA complex. The netropsin molecule is covered with foil for the sake of clarity. a, electrostatic interactions between ends of netropsin and DNA; b, hydrogen bond between netropsin and 2-keto of pyrimidine; c, $-hydrogen of adenylic acid (or inosinic acid), the position which hinders netropsin binding when guanylic acid replaces A (or I). Top, photograph taken at 90” to long axis of DNA helix. Bottom, photograph taken at approximately 45” to helix.

complex. Studies on the linear dichroism of netropsin-DNA complexes5 indicated only one orientation of netropsin, with respect to the DNA helix (the orientation of the absorption moment of netropsin is neither parallel nor perpendicular to the plane of the base pairs, but somewhere in between). The ADS of several natural DNAs revealed isosbestic points around 300 nm. Also, (B,,,)-r was the same for all netropsin-binding DNA polymers. Although these results do not prove that only one netropsin-DNA configuration dominates, they are all consistent with one complex. The binding of netropsin to (dG). and viscosity studies by Zimmer et al. (8) indicated the existence of other netropsin-DNA complexes, but these appeared to be weak and nonspecific. (b) The importance of electrostatic attractions between netropsin end groups and DNA phosphates, and the minor groove of DNA were discussed above. (c) From the com- parison of the allosteric model of ligand binding with the DNA

s M. Craig. J. Schellman, R. Wartell, and R. D. Wells, un-

polymers, a range of three base pairs per netropsin was found. The analysis of the natural DNA data indicated that several base pairs form a netropsin binding site. We interpret the range of three base pairs as the length of DNA covered per netropsin. (d) Netropsin showed no evidence of intercalation or unwinding of the DNA helix.

A molecular model of the netropsin-DNA complex was con- structed which accounts for these and other features (Fig. 11). The netropsin molecule is predominantly planar as a result of hyperconjugation.

This extended flat molecule bridges the two strands of the duplex DNA in the minor groove. The propionamidino and guanidino groups interact with phosphate oxygens on opposite strands of the same A-T pair (perhaps both on the 5’-side of the pair). Hydrogen bonds are formed between these basic groups and the phosphate oxygens of the A-T pair. This explains the

necessity for duplex DNA. The plane of the pyrrole rings (both in the same plane) is tilted about 30” from the plane of the base pairs. A hydrogen bond connects the amide NH (between the guanidino end and the first pyrrole ring of netropsin) and the 2- keto oxygen of thymidine. An alternate structure, in which the netropsin molecule is rotated 180” so that the two basic ends are transposed, may also be possible. This model explains the marked preference of netropsin for A-T (or I-C) over G-C, since the presence of a 2-amino group on G is too large to permit the close association of netropsin with the minor groove of DNA, and it disallows hydrogen bonding of the drug to the 2-carbonyl oxygen of the pyrimidine (either T or C). The model also ex plains the preference of netropsin for DNA duplexes over RNA duplexes, since it effectively serves as a spanner between the phosphate oxygens on opposite sides of the base pair. For RNA helices, this distance is appreciably greater than for DNA helices. It should be emphasized that this is only a preliminary interpretative structure which is consistent with the data.

Acknowledgment-We thank J. F. Burd for helpful discussions and for critically reading the manuscript.

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Appendix

Analysis

Assuming Various Netropsin Binding Sites

The theoretical model employed below was first Proposed by Crothers

(33) and considered in detail by Gurskii c &. (41r). A small mole-

cule, Nt in OUP case, binds in an all OP none fashion to the binding

sites of DNA. A binding site can be one or several base pairs which

are recognized by the molecule. There may be different types of

sites, each with a different binding constant K. When bound, every

Nt occupies ! . base Pairs. Each DNA base Pair can be occupied by

one Nt at nlost. It is assumed that transposition of the base pairs

(A-T or T-A) has no effect on binding. Gurskii c &. have examined

the Problem of ligand binding to a DNA with a heterogeneous nucleo-

tide sequence. We will follow their formulation and employ it to

obtain equations for various Nt sites.

In the asymptotic limit of r-r0 one has (Bap)-' and KaP BaP

defined as (See Fig. 3 and eq. (2) )

K B aP aP

= lim (r/Df) (A-la)

r-0

(B,J-l = lim a (r/Df)I ar

lim (r/Df) X-+0 (A-lb)

Gurskii z al. derived the relations

lim (r/D*) = (Z,} I N ZO (A-2=)

r-0

of disallowed states due to the overlap of nearby Potential sites.

From eqs. (A-l), (A-:a), (A-4) and (A-5)

(B,J1 = (1 + 2(P-1) CA) / CA (A-6s.)

K w BaP

= CA K1 (A-6b)

FOP two consecutive A-T* per Nt site

(Z,) = Z. N C; K1 (A-7a)

{ell) = NW; + 2 C; + C; 2(P-2) (A-7b)

The equations for the binding parameters follow from eqs. (A-l),

(A-?a), and (A-4)

(B&-l = (1 + 2CA + 2 (E-2) c:, / c: (A-b.1 e,2

K aP BaP

= c; K1 (A-Bb) _

For three consecutive AT pairs Per Rt site one finds in a manner

similar to the above

bl) = 3 '0 ' 'A % (A-h)

&) = N C; (1 + 2 CA + 2 C:, + 2 C; (E-3) ) (A-9b)

(1 + 2 CA + 2 c: + 2 c; (i-3) I c: (A-lOa)

123

B aP KaP

3 = CA K1 (A-lob)

(A-2b)

where Zi is the statistical sum of a DNA with i Nt molecules bound.

For a random base pair sequence DNA with AT content CA, averaging

is taken over all base sequences consistent with CA. The parti-

tion function Z2 is written by Gurskiic &. as

(A-3)

*ihere u is the number of different type binding sites, and Ku

the binding constant for site of type a. ma8 is the total number

of KaKB stares counted in 7.: / Zi which are disallowed because

the first bound Nt can occupy potential sites of the second Nt.

When there are two types of binding sites, v = 2, eqs. (A-2b)

and (A-3) yield

2; (a(rm,) I ar) = - c&J Kf + &JK~K~ +e2$K1~2 +422)K: 1

(23 / zo (A-‘+)

In the following calculations end effects will be neglected under

the assumption N )) L where N is the number of base Pairs per DNA.

If Nts binding site is one A-T Pair, then K2 = 0 in eq. (A-9).

and

'z \ 1) = z. N 'A K1 (A-5a)

N CA + N CA (2(t-1) CA) (A-5b)

Eq. (A-&S) counts a11 Potential binding sites of z single Nt. The

first term of eq. (A-5b) is the number of states disallowed because

two tit cannot occupy the same site. The second term is the number

A case of Nt interacting with two rypes of binding sites will

be examined next. A type 1 site will be designated as three

consecutive A-T pairs (AAAJ, and a type 2 site as two consecutive

A-Ts with a G-C to the left 0~ right (AAB OII BAA). The equilibrium

constant of a type 1 site, K1, is set equal to pK2. (Z1) and the

(Cij) are then given by

(Zl> = Z. N CC: p K2 + 2 C; CG K2) (A-lla) where CG= 1 - CA

($,;, = N C; (2 CG + 2 CA CG + ') C; CG (t-3) ) (A-lib)

621) = 2 N C: CG (CA + Cl + 2 C; (t-3) (A-11~)

2 2 ,'02*> T 2 N CA CG (1 + 4 CA CG (t-3) + CG + C; + CA CG) (A-lid)

113

(#,l) is identical to eq. (A-9b). Employing eq*. (A-l), (A-Za),

(A-4) and (A-11)

(Bap)-' = G P2 (p c; + 2 c; CGP

(1 + 2 CA + 2 c: + 2 c; (P-3) ) +

+ 4 p (CG + CACG + 2 c; CG W-3) ) + (A-12a)

+ 2 CG (CG $1 + CA1 + CA l CG + 4 CG CA U-3) )

K aP BaP

= (p C; + 2 C; CG) K2 (A-12b)

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Roger M. Wartell, Jacquelynn E. Larson and Robert D. WellsDEOXYRIBONUCLEIC ACID

Netropsin: A SPECIFIC PROBE FOR A-T REGIONS OF DUPLEX

1974, 249:6719-6731.J. Biol. Chem. 

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