Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

J. Mol. Hiol. (1984) 173, 63-74

Dipsticking the Major Groove of DNA with Enzymatically Incorporated Spin-labeled Deoxyuridines Electron Spin Resonance Spectroscopy ALBERT

M. BOBST, JOHN

University

SHIH-CHUNG

C. IRELAND

KAO,

AND INGRID

by

C. TOPPIN E. THOMAS

ROLAND

Department of Chemistry of Cincinnati, Cincinnati, Ohio 45221, CJ.S.A. (Received

10 June 1983)

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by elrctron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron’spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.

1. Introduction The sensitivity of spin-labeled nucleic acids to monitor conformational changes has been established in this laboratory as well as elsewhere (Bobst, 1972,1979,1980; Dugas: 1977; Kamsolova & Postnikova, 1981; Luoma et al., 1982). Here we use site-specifically spin-labeled nucleic acids to “dipstick” the major groove of a DNA duplex in a solution of low ionic strength. For that purpose, site-specifically spin-labeled deoxyuridine triphosphates with tethers of variable length were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer, which then was annealed with poly(dA). It is known that the structure of (dT),(dA), fibers in low ionic strength is similar to the structure of B-DNA (Amott & Selsing, 1974; Wells et al., 1977), and it is believed that the B-DNA fiber contains a wide major groove and a narrow minor one, both being of comparable depth and of the order of 8 A (Arnott, 1981). Based on our dipsticking experiments, we present evidence that the major groove of B-DNA in a solution of low ionic strength is of t’he order of 8 A, t’hereby corroborating X-ray spectroscopy data. 0022 -zx:~~/s4/or,oos~~--~~ to3.oojo

c 1984 Academic Press Inc. (London) Ltd.

.A 11 130I3ST E 7’ A 1,

64

2. Materials

and Methods

(dA), was purchased from P-L Biochemicals and was purified through Sephacryl S-200 before use. The Minit molecular building system was manufactured by Cocahranes of Oxford Ltd. Leafield, Oxford, England. OX8 5iVT. Analytical high-pressure liquid chromatography purity checks of the synthesized spin-labeled mononucleotides were performed on a Synchropak AX 300 column from Bynchrom. Inc. All other mat,erials were obtained commercially and were of analytical grade. (a) Synthesis

of (pppY”dl:),

5-Mercapto-2’-deoxyuridine-Striphosphate was synthesized according to a previously described procedure (Ho et al.. 1978). and isolated as the ammonium salt, with a yield of 20 to 30%.

(b) Preparation

of the r&oxide-containing

alkylating

agents

Epoxy tempo? was purchased from the Josef Stefan Institut in Yugoslavia. Activation of 4-(ol)-chloroacetamido-2,2,6,6-tetramethylpiperidino-l-oxy (Eastman) with sodium iodide in anhydrous acetone (Ozinskas & Bobst, 1979) yielded 4-(cc)-iodoacetamido-2,2,6,6tetramethylpiperidino-I-oxy. Reaction of 1.2 mmol of J-chloropropanoic acid (Aldrich) and an equivalent amount of 4-amino-2,2,6,6-tetramethylpiperidino-1-oxy (Eastman) in the presence of a 50/b excess of N,N’-dicyclohexyl carbodiimide (Aldrich) in dichloromethane at 4°C for 5 days, gave /I-chloropropanamido tempo in 82% yield after isolation from a preparative thin-layer chromatography plate (RF 0.40; methanol/chloroform, 1 : 19, v/v). Characterization: mass spectrum, m/z 261 for M+. The more reactive P-iodopropanamido tempo was prepared by reacting the chlorinated derivative with a 1.4 molar excess of sodium iodide in anhydrous acetone at 50°C for 2 days. y-Bromobutanamido tempo was prepared similarly to P-chloropropanamido tempo using 4-bromobutyric acid (Alfa) as the halogenated aliphatic acid instead of the 3-chloropropanoic acid. It was isolated as an orange oil in 26% yield. Characterization: mass spectrum. m/z 319. 321 (1 : 1) for M+. (c) Synthesis

of pppDCM

MT

To a stirred solution containing 0.016 mmol of (ppps’dU), in 0.2 ml of 0.5 N-KHzPO, were added a 15 molar excess of DTT (Sigma). After 30 min, 0.032 mmol of epoxy tempo, dissolved in 0.2 ml of acetone, were added to the reaction mixture. Similar amounts of DTT and alkylating agent were added as before at 2 h intervals. Aft,er a total of 6.5 h, the reaction mixture was streaked onto Whatman 3MM paper. The rhromatogram was developed with absolute ethanol/l M-ammonium acetate (7 : 3. v/v), and crude pppDUMMT was isolated after elution of the band of R,. 0.33 with water. Subsequent t Abbreviations used: (ppps’dU),, bis[l-(5’-0-triphosphono-~-o-2’-deoxyribofuranosyl)ur~~il-5-~l~ disulfide; epoxy tempo, 5,5,7,7-tetramethylpiperidino-6-oxy-2,Soxirane; b-chloropropanamido tempo, 4-(~)-chloropropanamido-2,2,6,6-tetramethylpiperidino-l-o~y; j-iodopropanamido tempo, 4-(j)iodopropanamido-2,2,6,6-tetramethylpiperidino-l-oxy; y-bromobutanamido tempo, 4-(y)-bromobutanamido-2,2,6,6-tetramethylpiperidino-I-oxy; (DUMMT,dT),, copolymer of DUMMT and thymidine; DUMMT, [(l-oxyl-2,2,6,6-tetramethyl-4-hydroxy-4-piperidinyl)methyl]-(l-~-D-2’~ deoxyribofuranosyluracil-5yl)-sulfide: (DUTT,dT),, copolymer of DUTT and thymidine; DUTT, [N-(1-oxyl-2,2,6,6-tetramethy~-4-piperidinyl)methylcarbamoyl]-(l-~-D-2’-deoxyribofuranosyluracil-5-yl)-sulfide; (DUMPT,dT),, copolymer of DUMPT and thymidine; DUMPT, [N-( l-oxyl2,2,6,6-tetramethyl-4-piperidinyl)ethylcarbamoyl] (1 -,Co-2’.deoxyribofuranosyluracil-5.~1) -sulfide: (DUMBT,dT),, copolymer of DUMBT and thymidine; DUMBT, [N-(1-oxyl-2,2,6,6-tetramethyl4-piperidinyl)propylcarbamoyl]-( 1-/-n-2’deoxyribofuranosyluraciWyl)-sulfide: DTT, dithiothreitol; t$r’, melting temperature determined by ultraviolet light spectroscopy; t,, and rI, correlation times for rotations about and perpendicular to the principal axis of diffusion; n.m.r., nuclear magnetic resonance; e.s.r., electron spin resonance.

I)IPSTICKIS(:

OF THE

MAJOR

DNA

6.5

GROOVE

repurification on a DEAE-Sephadex column (15 cm x 16 mm) eluted with a linear gradient of 0.1 .n to 0.35 M-NH~HCO~ gave pure pppDUMMT as judged by high-pressure liquid chromatography. ‘H n.m.r. analysis of the %monophosphate of DUMMT in 2H20 after reduction with sodium dithi0nit.e (Ozinskas & Bobst,, 1980). with tetramethylammonium chloride as standard (3.0 S), confirmed the nucleoside structure and gave the following chemical shift values: C6-H, 8.1 6 (5); CH, (exocyclic), 2.7 6 (s); CH, (piperidine), 18 6 (m); CH, (piperidine), 1.2 6 (s) and 1.5 6 (s); W-H. 6.1 6 (t): C2’-H. 2.2 6 (m): C3’-H, 4.5 6 (s): C4’-H. 3.9 6 (s) and (X-H, 4.0 6 (s). (d) The procedure for the preparation

of pppD7.1’7

Synthesis

of pppDUTT

has been published (Toppin et al., 1983).

of pppDC’MPT

(e) Synthmis

Reaction of (ppps5dU)2 with b-iodopropanamido tempo and DTT, under conditions similar t’o those used to obtain pppDUMMT. gave pppDUMPT in 5O”/b yield. The ‘H n.m.r. chemical shift values of t,hr reduced 5’-monoph0sphat.e of DUMPT are: C6-H, 8.0 6 (s); C’H, (exocyclic, adjacent to sulfur). 2.7 6 (m); CH, (exocyclic. adjacent to carbonyl). 2.4 6 (m); CH (piperidine), 2.9 6 (m): CH, (piperidine). I.8 6 (d): CH, (piperidine). 1.2 6 (s) and 1.3 6 (w): Cl’-H. 6.1 6 (t); (Y--H. 2.2 6 (m) and C3’-H to C5’-H. 3.7 6 to 4.5 6 (m). (f)

of pppDl’MB1’

Synthesis

(ppps5dU), was alkylated with y-bromobutanamido tempo after reduction wit,h DTT using the same procedure as described for pppDUMMT. Approximately 50% conversion of nucleotide disulfide to pure pppDUMBT (as judged by high-pressure liquid chromatography) was observed. The ‘H n.m.r. chemical shift values of the reduced 5’-monophosphate of DUMBT are: C66H. 7.9 6 (s): CH, (exocyclic. adjacent to sulfur). 2.66 (t)): CH, (exocyclic. adjacent to 2 methylene groups). 2.1 6 (t) and 2.3 6 (t); CH, (rxocyrlic, adjacent to carbonyl). 1.7 6 (t): CH (piperidine). 1.8 6 (m): CH, (piperidine), 1.86 (m): CH, (piperidine). 1.26 (s) and 1.36 (s): Cl’-H. 6.1 6 (t): (2-H. 2.26 (tn) and C%H t,o (5-H. 3.8 6 to 4.4 6 (m). of

(g) Incorporation

spiwlabeled

2’-deoxyuridine

analogs

into

DA’A

The conditions for the copolymrrizat~ion of pppDUTT with thymidine 5’-triphosphate by terminal deoxynucleotidyl transferase have been described (Toppin et al., 1983). Using similar reaction conditions but. substituting pppDUMMT. pppDUMPT and pppDUMBT for pppDlrTT led to the production of the spin-labeled copolymers (DUMMT,dT),. (DUMPT,dT), and (DCMBT.dT),, respectively. All spin-labeled single strands were isolated and characterized similarly to (DUTT.dT), (Toppin et al., 1983) and those used for this study had a weight average molecular weight of 100,000 to 200,000 and contained 1 to 296 of spin-labeled nucleotidrs. (h) LVW&YWmagnetic

reSonance

spectroscopy

The n.m.r. spectra were obtained on a Nicolet XTC 300 FT Instrument and the electron impact mass spectra on a Perkin Elmer RMU-7 spectrometer. A Gilford 250 spectrophotometer and a Gilford 2527 thermoprogrammer using a cell assembly consisting of 4 quartz microcells in an electrically heated block at a heating rate of 1 deg. C/min was used to determine the ultraviolet absorbance thermal drnaturation profiles. (i) to

Electron

spin

resonance

spectroscopy

All e.s.r. spectra were measured with a Varian E-104 spectrometer interfaced according Trrland rt al. (1983) to an Apple IT plus microcomputer. The e.s.r. simulations were

lili

.\

hl. 1~Ol~S’l‘ E7’ ‘1 I,

cwrird out on an Amdahl 470 computrr with a tilt program used hy Mason rt al. (1974) whic,h, for the present analysis has heen modified to include odd 1, states (Polnaszrk. 1976: Kao rt CL/.. 1983). The simulation program takes into acacaount anisot,ropic rotational motion. i.e. the rotational motion of the molrculr is czharactrrizrd h\- 2 cwwlation times. z for ahout the 2 orthogonal motions about the principal axis of’ diffusion z’. and TV f’or mo&ns diffusion axes of the molewlr. The simulation program and thr motional model used hrrr have hren discussed in more detail elsewhere (Kao r+ al.. 1983). Thr rxprrimt~ntal and simulated spectra were plotjtrd on a Houston Tnstwmrnt DMT’-3 digital plott~er.

3. Results In

Figure

deoxyuridines

I the chemical structures of the four &substituted spin-labeled are shown. The length of the tether increases hy two honds from

dr

DUMMT

R=S

DUTT

DUMPT

0

I+:.

I:c

I. Muhntituted

N-O

spin-labeled deoxyuridines.

DUMBT

DIPSTICKISU

OF THE

MAJOR

DSA

GROOVE

(ii

DUMMT to DUTT, by an additional bond from DUTT to DUMPT, and by one more from DUMPT to DUMBT. In all cases, the tether connects the sixmembered nitroxide ring via a sulfur linkage to the deoxyuridine. The pyrimidine sulfur linkage is used as the principal rotational diffusion axis Z’ for the e.s.r. simulation calculations (Kao et aE., 1983). A model of the (DUMBT,dT),(dA), d u pl ex with the spin label DUMBT is displayed in Figure 2. The six-membered nitroxide ring can be seen on the right half of the model and the size of the leg, protruding into the major groove, is clearly visible. Reducing the leg size results in bringing the six-membered nitroxide ring deeper and deeper into the major groove (not shown). In Table 1 the melting properties of the spin-labeled duplexes (DUMMT,dT),(dA),, (DUTT,dT),(dA),, (DUMPT,dT),(dA), and (DUMBT,dT), (dA), are summarized. As already noticed earlier for (RUGT,U),(A), (Langemeier Br Bob& 1981), the thermal stability of t’he duplex is not affected by the presence of the spin-labeled nucleotides when the subst’itution is in position 5 of the pyrimidine and the amount of spin incorporation into the nucleic acid lattice is of the order of a few per cent. The experimental and simulated e.s.r. spect’ra of the single-stranded (dT), derivatives (DUMMT,dT),, (DUTT,dT),, (DUMPT,dT), and (DUMBT,dT), are shown in Figure 3. The e.s.r. lineshapes are similar for all and reflect motional narrowing region spectra. Based on the peak heights of the high field lines, it can be concluded qualitatively that the motion of the nitroxide radical is slightly more hindered in (DUMMT,dT), than in (DUMBT,dT),. The e.s.r. simulations were carried out as described for (RUGT,U), (Langemeier & Bobst, 1981), but were improved by introducing a tilt angle between the diffusion and magnetic axes: which is defined as follows: One of the axes for TV is chosen coincident with the magnetic y axis and the principal axis of diffusion z’ initially coincides with the magnetic z axis. The diffusion axis Z’ can then he rotated about the y axis by a tilt angle (see Fig. 1 of Robinson & Dalton, 1980). Therefore, the present program requires one additional input parameter, the tilt angle, as compared to that used earlier. Tt is similar to the program given by Mason et al. (1974), but has been to modified to include odd L states (Polnaszek, 1976) and was used recently analyze spin-labeled RNA systems (Kao et al., 1983). The parameters used for the computer simulations of the spin-labeled single strands are given in Table 2. A tilt angle of 55 (15)” was used for the simulation of all single-stranded systems, except for (DUMMT,dT),, where a slightly smaller angle was used, possibly reflecting the effect of the additional hydroxide group in the six-membered ring of the DLTMMT label. T,,,which is believed to represent the motion of the tether axis. increases from 0.03 to 0.09 ns as a function of decreasing tether length, whereas TV is of the order of 1.2 ns for all single stranded systems. Figure 4 shows the experimental and simulated e.s.r. spectra of the double st.rands (DCMMT,dT),(dA),, (DUTT,dT),(dA),, (DUTMPT.dT),(dA), and (DUMtHT,dT),(dA),, together with the chemical structures of the probes. The tlramat,ic effect on the e.s.r. lineshape caused by increasing the tether length of one of the probes by one bond is rlearly observable. Samely. as long as the tether does not exceed a critical length, the e.s.r. spectra of the duplexes remain

in a. H-LJX.4 form FIG. 2. Minit molecular model depicting (DIIMBT.dT),(dA). ;tttac~hrd in position 5 of the pyrimidinr base and protrudes into the major yrwrr c,rdinatrs of H-I)NA wvpre obtained from .\rnott & Hukins (1972).

‘I’hv spm Iahrl ih ‘I’hr atomic vow

DIPSTICKIN(:

OP THE

MAJOR

TABLE

DNA

GROOVE

ti9

1

Effect of spin label in position 5 on ti” of (dT),(dA), Double-stranded

t”,” ("C)

system

(dWdA)n

54 * 0.5 54+0.5 54 * 0.5 54 + 0.5 54 It 0.5

(DUMMT,dT),(dA), (DITTT,dT),(dA), (DUMPT,dT),(dA), (DI;MBT,dT),(dA),

broadened and are not too different from one another. However, after the critical length is reached, the addition of one more carbon unit results in an e.s.r. spectrum with narrow lines, which is a definite indication of greater mobility. The e.s.r. simulations were achieved with the parameters listed in Table 3. zI is about 4 ns for all duplexes, whereas z,, displays a dependence on tether length. The tilt angles are similar for the DUTT and DUMPT-containing duplexes and are

(=-+AdfIO G

Fro. 3. Experimental (---) and computer-simulated (-------) e.s.r. spectra of spin-labeled single strands. The experimental spectra were taken in 0.01 h!-NaCl, 0.01 M-sodium cacodylate (pH 7). (a) 1.13 x 1O-4 rv-(DUMMT,dT),; (b) 3.9 x lo-’ w(DUTT,dT),; (c) 4.4 x lo-’ M-(DUMPT,dT),; (d) l-4 x 10-4 M-(DUMBT,dT),. The computer-simulated spectra were obtained as described in the text, with the parameters given in Table 2.

Single-stranded

system

(DUMMT,dT), (DIJTT,dT), (DlJMPT,dT)” (DUMHT,dT),

Tll (ns)

Tl (ns)

Tilt angle (deg.)

04!) wo7 046 043

I.2 1.2 1.2 I.2

45 55 55 .i:‘,

Nitroxide e.s.r. parameters: A,, = 7.15 Gauss; A,, = 7.35 Gauss; A,, = 356 Gauss; gxx = 2W88; y,, = 290.59; gzz = 2.0026. Additional line broadening (T;‘): 0.8 Gauss for (RUMMT,dT),; 1.2 Gauss for (DUTT,dT),. (DCJMPT,dT), and (DUMBT,dT),.

FIN. 4. Experimental (--) and computer-simulated (-------) e.s.r. spectra of the spin-labeled double strands with the chemical structures of the probes. The experimental spectra were taken in 0.01 M-NaCl, 0.01 M-sodium cacodylate (pH 7). (a) 2.26 x 10m4 M-(DUMMT,dT),(dA),; (b) 7.8 x IO-’ M(DIJTT,dT),(dA),; (c) 8.8 x 10-s M-(DUMPT,dT),(dA),; (d) 2.8 x 10e4 M-(DUMBT,dT),(dA),. The computer-simulated spectra were obtained as described in the text, with the parameters given in Table 3.

DIPSTICKING

OF THE

MAJOR

TABLE ISffect

qf tether

length

of spin

label

Double-stranded

system

(l>UMMT,dT),(dA), (DUTT,dT),(dA), (DUMPT,dT),(dA), (DUMBT,dT),(dA),

UROO\‘E

71

3

on the motional

DNA

DXA

parameters

of the double-stranded

systems

?I1(ns)

TI (ns)

Tilt angle (deg.)

0.7 0.3 0.2 0.06

4 4 4 4

80 40 40 55

Nitroxide e.s.r. parameters for (DUMMT,dT),(dA),: A,, = 7.35 Gauss: A,, = 3545 Gauss; A,, = 7.15 (:auss; grx = 2.0059; gvu = 2.0026; gzz = 2+W38; additional line broadening (T;‘): 0% Gauss. Parameters for (DlTTT,dT),(dA),, (DUMPT,dT),(dA), and (DUMBT,dT),(dA),: A,, = 7.15 Gauss; A,, = 7.35 Gauss; A,, = 35% Gauss; gxx = 2.0088; qYY= 2.0059; gzz = 2.0026; additional line broadening (TF ‘): 0.8 Gauss for (DUTT,dT),(dA), and (DUMPT.dT),(dA),; I.0 Gauss for (I)I:MI~T.ti’r),(dA),.

both a little smaller than in (DUTT,dT), or (DUMPT,dT),. In the case of DUMMT, which is closest to the base, complex formation results in the largest tilt angle change, of the order of 35”. On the other hand, DUMBT gives the same tilt angle in both the base-paired and non-base-paired configuration. Furthermore, a comparison of the values in Tables 2 and 3 for the DUMBT-containing nucleic acids indicates that the DUMBT in the duplex requires similar simulation parameters as when non-base-paired, except for zI, which is believed to reflect the base motion and remains the same for all base-paired systems.

4. Discussion A CPK space-filling model of a spin-labeled D?\‘A duplex shows that the various spin labels, when attached in position 5 of the pyrimidine base, will protrude into the major groove of the duplex. The Minit model, as shown in Figure 2, suggests that none of the three shorter tethers is likely to fold back on itself, due to steric hindrance arising from the bases and sugar backbone as well as due to their inherent rigidity. A folding back of the longest leg cannot be ruled out, but does not seem to occur or else the nitroxide ring would have remained trapped in the major groove and the sudden change in mobility would not have been observed. Thus the tethers seem to behave as real dipsticks. A simple geometric model describing the position of the spin label in the major groove is proposed, which allows a correlation between tether length and the e.s.r. lineshapes. As long as the nitroxide ring remains within t’he major groove. a hindered motion with a broadened e.s.r. lineshape should be expected. On the other hand. if the nitroxide ring reaches beyond the cavit’y, its motion will be increased significantly. Based on this model, and in view of the data shown in Figure 4, the longer tether of DUMBT is required so that the nitroxide ring motion is no longer hindered by the cavity. This simple model can be further expanded with the diagram shown in Figure 5. which is used to understand t)he rapid motion of the bases. which seems to occur

FIG. 5. Schematic diagram of DUMPT 2 axis is tilted by an angle with respect of the nitroxide ring are not shown.

base-paired with dA. The molecular fixed hyperfine principal to the principal rotat,ional diffusion axis 7’. The methyl groups

in the 4 ns time-frame. First,, it should be pointed out that other motions, for instance the rotation of the spin-labeled DNA duplex as a whole, could contribut,e to the relaxation of the nitroxide. Based on hydrodynamic considerations, a mean correlation time of 200 ns was calculated for a DNA molecule of about 550 basepairs rotating uniformly (Robinson et al.. 1980). The present spin-labeled duplexes, which consist of about 300 base-pairs, give a correlation time of about’ 100 ns with the same calculation approach and, therefore. the rotation of the spinlabeled DNA duplex as a whole can be neglected from the motional model illustrated with Figure 5. The diagram in Figure 5 shows DUMPT base-paired with dA. One of the axes for zL is chosen to coincide with the magnetic y axis, and the principal axis of diffusion 2’ is initially coincident with the magnet’ic z axis, but then can be rotated about the y axis by the tilt angle (see also Fig. 1 of Robinson & Dalton, 1980). rL can arise from torsion and tilting of the base-pairs and twisting of the bases as well as rotation of bond 2 or any bond parallel to it. However, the latter motion does not seem to be included in z,, since rL was found to be constant and of the order of 4 ns. On the other hand, z,, is dependent on tether length. As long as the nitroxide remains in the major groove, z,, decreases slowly with increasing tether length, but then undergoes a substantial decrease from (DUMPT,dT),(dA), to (DUMBT,dT),(dA),. The fully extended tether length, separating the nitroxide ring from the pyrimidine base in the case of DUMPT is calculated to be 7.5 A with standard bond lengths and bond angles, whereas for DUMBT a value of 9 A is obtained. For that reason, we conclude that the depth of the major groove in a (dT),(dA), duplex is of the order of 8 A in a solution of low ionic strength. This finding for the depth of the major groove of a DNA system in solubion is in good agreement, with measurements done on DNA fibers by X-ray spectroscopy. which give a value of 8.5 A (Arnott. 1981). Furthermore, using tet’hers of various lengths together wit*h the present model for the interpretation of the experimental e.s.r. data allows one to uncouple the motion of the base from the spin label motion. We have reported preliminary e.s.r. data for (DUTT,dT),(dA), (Bobst et al., 1981), but at that time we were

DIPSTICKING

OF THE

MAJOR

DNA

GROOVE

73

unable t,o evaluate the mobility contribution of the tether. Also, the simulations carried out earlier did not fit the experimental data as well, because the anisotropic model did not include a tilt angle between the diffusion and magnetic axes. The values for T,, and 51 were found to be 1.1 x 10m9 s and 2.3 x lo-’ s, respectively, and are in qualitative agreement with the values reported in Table 3. It is interesting to note that we determine base motions wit’h the present spin labeling approach that’ are about a factor of ten faster than those reported most recenGy in the literature (Hogan et nl., 1982: Hurley et al., 1982). Tn conclusion, we show that macromolecular spin probes obtained after site-specifically deoxyuridine enzymatic incorporation of spin-labeled triphosphates have potential as sensitive hybridization probes. since hybridization with some of the probes will cause well-characterized and significant e.s.r. lineshape changes. Also, using a set of different, tethers on the spin-labeled nuclrosides allows one to determine the depth of grooves in nucleic acid systems and to conclude that the mobilit? of the bases in a D?iA duplex has a correlation time of t,he order of nanoseconds. This investigation was supported in part by grants from U.S. National Science Foundation (No. PCM 7801979) and by U.S. Public Health Service (No. GM 27002). J’urchasc of the Nicolet NTC 300 FT Instrument was supported in part by National Scirncbc,Foundation grant CHE-81-02974. REFERENCES Arnott. S. (1981). In Topics in A%&& Acid Structurr (Neidle, S.. ed.), pp. 65-82, *John Wiley & Sons, New York. Arnot,t. 8. &, Selsing, E. (1974). J. Mol. Biol. 88, 509-521. Arnot,t, S. & Hukins, D. W. L. (1972). Biochem. Biophys. Res. Commun. 47, 1504&1509. Kobst. ,4. M. (1972). BiopoZymers, 11, 1421-1433. Bob&, A. M. (1979). In Spin Labeling ZZ: Theory and Applications (Berliner. I,. J.. ed.). pp. 291-345, Academic Press, New York. Bob&, A. M. (1980). In MoZecular Motion in PoZymers by ESR (Bayer, R. F. & Keinath. S. E.. eds), pp. 167-175, Harwood Academic Publishers, New York. Bob&. A. M., Ireland, J. C. & Langemeier, P. W. (1981). Biophys. J. 33. 313a. Jjugas. H. (1977). ilcc. Chem. Res. 10, 47-54. Ho. Y. K., Novak. L. & Bardos, T. ,J. (1978). In Nucleic Acid C’hemistry (Townsend, S. B. B Tipson, R. S.. eds), part II, pp. 813-816, John Wiley and Sons, New York. Hogan. M. E.. Wang, J., Austin, R. H., Monitto, C. 1~.& Hershkowitz, S. (1982). Proc. IVat. Acnd. Ski., U.S.A.

Hrtrlry.

79, 3518-3522.

1.. Osei-Gyimah,

Bioch~emiatry,

P., Archer,

S.. Scholes, (1. I’.

& Lerman.

L. S. (1982).

21, 4999%5009.

Trrland, J. CT..Willett, ,J. A. & Bobst, 4. M. (1983). J. B&hem. Biophys. Methods, 8, 49-56. Kamsolova. S. G. & Postnikova, G. R. (1981). & uart. Rev. Biophys. 14, 223S288. Km. S. (1.. Polnaszek, C. F.. Toppin, (J. R. & Bobst. A. lf. (1983). Biochemistry, 22, 55635568. Langrmrir. I’. W. & Bobst, A. M. (1981). Arch. Biochem. Biophys. 208, 205-211. Luoma. (:. A., Herring, F. G. & Marshall, A. G. (1982). Biochemistry, 21, 6591-6598. Mason. R. I’.. Polnaszek, C. F. & Freed, J. H. (1974). J. Phys. Chem. 78, 1324-1329. Ozinskas. A. .J. & Bobst, A. M. (1979). HeZw. Chim. Acta, 62, 1677p1681. Ozinskas, A. ,J. & Hobst, A. M. (1980). Helw. Chim. Acta. 63. 140p141 1. Polnaszek. (“. F. (1976). Ph.D. thesis, Cornell University. Robinson. I<. H. & Dalton. L. R. (1980). J. Chem. Phys. 72. 1312-1324.

71

.A 11 I3 0 13ST E7 .-I /,

Robinson, K. H.. Lermam. 1,. S., IMh. A. H.. Frisch. H. I,., I)alton, 1,. K. & Auer, 1’. (l!M)). J. Mol. Hid. 139. 1%41. Toppin, (‘. R.. Thomas. 1. E.. Kobst.. E. v. 8: Bobst, .L\. ib1. (1’383). Int. ./. &ol. :bffrcromo/. 5.

33

-36.

Wrlls, R. I).. Blakesley, R. W., Hadies, S. C’.. Horn, C. T.. Larson. ,J. E.. Selsing, E.. Bud, .J. F.. Ghan. H. W.. 1)otlgsop. ,J. B.,
Ih.

Hiochern.

4. 305-340.

Edited by M. Gellert