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Lanthanide ion luminescence as a probe of DNA structure. 2. Non-guanine-containing oligomers and nucleotides
Lanthanide Ion Luminescence as a Probe of DNA Structure. 2. Non-Guanine-Coming Oligomers and Nucleotides Scott L. IUakamp* and William Dew. Horrocks, Jr. Department Pen~yivania
of Chemistry,
The Pennsylvania
State
University,
University
Park,
ABSTRACT Oligo(dC),, oligo(dA),,
and oligo(dT& as well as d-CMP, d-AMP, and d-TMP, when complexed to ELI’+, possess two classes of Eu 3+ binding enviroument. The binding environments consist of two classes, tight sites which coordinate two Ha0 molecules, aud weaker sites which coordinate six or seven, analogous to the previously studied guanine-containing molecules. It is inferred that the tight class of Et?+ ion site observed with these oligomers and nucleotides corresponds to dimeric or polymeric structures. Comparison of the results for the guanine and ~-~~~ ~~~ ohgomers suggests that Eu3+ possibly coordinates base nitrogen atoms in the former and in an outer sphere mode (hydrogen bonding via the H,O molecules coordinated to Eu3+) in the species examined here.
In this paper the molecules oligo(dC),,
oligo(dA),, digo(dT),, d-CMP, d-AMP, and d-TMP are investigated by Eu3+ luminescence spectroscopy using the methods described in the preceding article [ 11. The major reason for undertakingthese studies was to determine if the base composition of DNA has any bearing on how Et?+ binds to single stranded DNA. It was suggested [l] that the N-7 ~~gens and/or O-6 oxygens of guanine could possibly coordinate to Eu3+. The base adenine has a N-7 nitrogen atom also, but no O-6 oxygen atom. The N-7 of adenine is also very weakly basic (pK, = - 1.6) [2], while the N-l of adenine is a much more basic site. The base cytosine has a carbonyl oxygen atom at O-2 and a nitrogen atom at N-3, while the base thymine possesses no unprotonated nitrogen atoms at pH 6.0. Transition metal ions favor coordination at N-3 of pyrimidine bases, and at the N-7
Present address, Division of Chemistry and Chemical Eng~~, California Institute of Technology, Pasadena, California. Address reprint requests and correspoudence to: W. D. Horrocks, Jr., Department of Chemistry, 152 Davey Lab, University park, PA 16802. Journal of Inorganic Biochemhtfy, 46, 193-205 (1992) 193 @ 1992EkevierSciencepublishingCo., Inc.. 655 Avenue of the Amnicas, NY, NY lOOtO Ol~~l~/~/$S.~
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S. L. Klakamp and W. DeW. Horrocks, Jr.
and N-l sites of purine bases [2]. Controversy exists in the literature [3-61 over whether Tb3+, or by analogy, Eu3+, coordinates-to the N-7 of guanine residues in addition to the commonly accepted phosphate oxygen coordination. Our study of the B-mers of the four DNA bases was carried out in order to provide insight into the role of the base in Ln3+ bi~ng. axon of the nucl~des allows a closer inspection of the monomeric units of which the 8-mers consist, and provides data which suggest that the Eu3 + complexes of d-CMP and d-AMP form dimeric structures in solution. These are the first studies to look directly at Ln3+ ion coordination to non-guaninecontaining DNA. MATERIALS AND METHODS Materials The o~g~~x~ucl~ti&s were purchased from Pharmacia, Inc. and used as supplied. All the nucleotides studied were purchased from Sigma Chemical Co. and used without further purification. Concentrations were determined spectrophotomet&ally for the studied biomolecules (with lot numbers in parentheses) using the following molar absorptivities at 260 nm: pd(C), (00815-114), 7200; pd(Q (~107~), 7800; pd(A)s (~35) 12,400; d-CMP (128C-7000), 7500; d-AMP (128C-7030), 15,400; d-TMP (63f-7000), 9200. All oligomers and nucleotides were dissolved in 20 mM NaCl and 20 mM MES buffer (PH 6.0) made up in doubly distilled H,O or D,O (Aldrich Chemical Co., 99.8%). EuCl, * 6H,O and ThCl * 6H,O were obtained from Aldrich Chemical Co. and used as supplied. Eu 3, solutions were standard&d by a chelometric method 173. Eu3 + Time-Resolved Luminescence Titrations The details of the laser experiments are identical to those described in the preceding paper [ 1J. Luminescence decays at each point in a titration were collected for 15 min (9000 transients) and corrected for fluctuations in laser power during the course of an experiment, The titrations of the oligomers were performed in D,O, and the time-resolved titrations of the nucleotides were done in H,O. Luminescence decays were analyzed as described in the preceding article [l]. The lifetimes of d-TMP, d-AMP, d-CMP, d-GMP, pd(C&,, and pd(T)s were held constant in the Marquardt analysis of their respective fits. The lifetimes determined for the pd(A)s system changed sig~fi~~y after one equivalent of Eu3+ ion was added, and therefore two sets of lifetime values, one set before and one set after the addition of one equivalent, were held constant in the nonlinear regression analysis (as described in the legend to Fig. 4). In the Marquardt analysis of d-TMP, one lifetime component was held constant at 110 ps. When all parameters were allowed to ride freely in the nonlinear regression analysis for d-TMP, no two lifetime components values were obtained consistently from a double exponential fit. Upon holding one lifetime component constant at 110 ps in the Marquardt analysis, a very consistent 165 ps lifetime value was generated, as shown by the standard deviation for this component in Table 1. The lifetime value of 110 ps was a logical guess (not arrived at by an average of lifetime values as in the other systems) for the second lifetime component, since single exponential analyses of the luminescence decay curves for d-TMP gave a lifetime value of 119 ps with very poor fits to the decay curves. The non-time-resolved titrations with Eu3+ were performed identically to those described previously [ 11.
L A N T H A N I D E ION PROBES O F D N A S T R U C T U R E . 2.
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TABLE 1. Determination of the Number of Water Molecules, q, Coordinated to EU3+ Bound to non-guanine-containing Oligomers and Nucleotides Oligomer pd(A)sc [Eu3+ ] < [DNA-P] [Eu3+ ] ~e [DNA-P] pd(T)sc pd(C)sc d_AMPc d_CMPc
"H 20(/~s) a
• D20(/zs)a
qb
311 + 10 136 + 5 311 5:10 136 + 5 446 5:35 143 5:6 333 5:26 155 5:17 350 5:16 115+4 473 5:95
578 + 41 2313 5:90 1025 + 62 2972 + 37 2993 + 76
2.0 + 0.5 7.3 + 0.5 2.1 + 0.5 7.4 5:0.5 2.8 5:0.5 6.4 + 0.5 1.8 + 0.5 6.4 + 0.5 2.7 + 0.5 8.8+0.5 1.9 + 0.5 8 . 5 + 0.5 6.0 5:0.5 9 . 2 + 0.5
761 5:104 2850 5:43 3198 + 118 3038 5:38
119 + 3
d_TMPc
165 5:16
2972 5:22
110 'q
a Errors shown are the standard deviation associated with each lifetime measurement. b Errors are ~ t e d as in D. Skoog and D. West, Fundamentals of Analytical Chemistry, Saunders College Publishing, Philadelphia, 1982, Chapter 3, pp. 39-82. c Eu3+ oligomer and nucleotide concentrations are those employed in their respective time-resolved titrations (Figs.3-6). dNo standard deviation is assigned because 110 ~ts is an estimate.
RESULTS N o n - T i m e - R e s o l v e d T i t r a t i o n s o f p d ( A ) e, p d ( C ) 8, a n d p d ( T ) s The excitation spectra o f the oligomers and nucleotides studied here are shown in Figure 1. The spectra o f the oligomers are very broad and similar to those o f the guanine-containing oligomers. In the non-time-resolved and time-resolved titrations an excitation wavelength o f 579.20 nm was used to eliminate, as much as possible, the contribution o f Eu 3+ aqua ion luminescence to the observed signal. The excitation peaks for the nucleotides are narrower than those o f the oligomers, consistent with the smaller number o f Eu 3 + environments capable o f being adopted by the nucleotide complex. A s seen in Figure 2, the curve for pd(T) 8 titrated with Eu 3+ breaks at 0.35 [ E u 3 + ] / [ D N A - P ] . The increase in luminescence beyond this point is largely due to that o f the aqua Eu 3 + ion. It appears that three phosphate moieties are coordinating per Eu 3+ in o l i g o ( d T ) s - - t h e number that would be expected based on charge neutralization considerations. However, evidence exists, which will be discussed later, for a possible intermolecular interaction (aggregate) between or among Eu3+-pd(T)s oligomers. The extent o f aggregation seen with lxl(T) s is, however, less than that with the guanine-containing oligumers. In any case, Eu 3+ binds PriG')8 strongly until 0.35 equivalents o f Eu 3+ have been added, which corresponds to the charge neutralization point. This is the only 8-mer investigated which exhibits this expected type o f behavior. The non-time-resolved titration (Fig. 2(13)) o f pd(C)s with Eu 3+ indicates behavior shnilar to that seen in the titration o f pal(G) 8 with Eu 3+ (Fig. 2 in preceding paper). The titration curve for lxi(C) 8 shows that Eu 3+ is bound tightly to pd(C) s until - 0 . 5 to 1.0 [ E u 3 + ] / [ D N A - P ] . The similarity seen between the pd(C) 8 and 1x1(O)8 titrations lies in the fact that both curves continue to indicate Eu 3+ binding even up
1%
S. L. KIakamp and W. De W. Homocks,Jr,
I
578.5
57s UAELENSTII
570
sm.6
sn UAbQ.MTH
m.5
SW
580.5
km)
08.5
m
911.6
Cd
FIGURE 1. 7FO-+‘D, excitation spectra (h
= 614 mu) of Eu3+ bound to various nucleotides and oligonucleotides. (Top panel): A: oligo(dC), (26 PM DNA-P); B: oligo(dA)8 (33 FM DNA-P); C: oBgo(dT)~ (27 pM DNA-P) at 1.0 ~~+]/[DNA-P]. (Bottom panel): A: d-TMP (35 HM); B: d-AMP (38 CM); C: d-CMP (33 FM) at 1.0 @h3+]/DNA-P].
to five added equivalents of Eu 3+. The slope of the line produced by the simple
addition of Eu3+ to buffer is less than the slope of the pd(C), titration curve from one to five equivalents. The increase in luminescence from one to five [Eu”+]/ [DNA-P] rest&s from the following three factors: 1) Eu3+ aqua ion luminescence, 2) additional biing of Eu3+ to pd(C), (single-stranded or aggregate), and (3) aggregation (crosslinking) of pd(C)s oligomers via Fu3+ ions bound to pd(C)s. In this last case, Eu3+ luminescence would increase owing to the dehydration of the inner coordination sphere of Eu3 + ions by eliminating a deexcitation pathway. nl~~ also in Fig. 2(C) is the non-~-~lv~ ti~ti~ of pd(A), with Eu3+. This curve is strikingly different from the curves for the other El-mers. Eu3+ ion coordinates pd(A)s tightly until [Eu3+],‘[DNA-P] = 1.0. The sharp increase in luminescence observed after one equivalent of Eu3+ may be explained by the more pronounced ability of pd(A)s to aggregate at lower concentrations of Eu3+. This is
LANTHANIDE
100 A
I
ION PROBES OF DNA STRUCTURE.
I
I
I
2.
197
Im
D
7sm D
m
w-
m
m mm0 .
2s mm D 0, 0
. I I
I 2
I ¶
I 4
I 6
6
1 6
CEUCIII>J/CDwI-PJ
FIGURE 2. Non-time-resolved titration with Eu3+ lumiuescence ()Lx = 579.20 um, &,,, = 614 run) plotted as a function of added Eu3+. A: pd(‘l& (0) (31 pM DNA-P in D,O, 20 mh4 NaCl and 20 mM MES, pD 6.0). B: pd(C), (0) (33 pM DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0). C: pd(A), (0) (31 CM DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0). (A) the simple addition of Eu3+ into buffer.
198
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supported by the turbidity observed for some pd(A)s solutions which was detected visually after the addition of - 4 to 5 equivalents of Eu3+. Additional data suggesting this stronger tendency towards aggregation is provided by an (A32O/A260) ratio of 0.18 at 3.0 [Eu]/[DNA-P] for pd(A), versus values of this ratio of 0.009 and 0.096 for pd(T), and pd(C),, respectively. Time-Resolved Eu3 + Luminescence Titration Studies The time-resolved titration of pdQ, with Eu3+ in D,O (Fig. 3) consists of a single exponential and, not surprisingly, corresponds closely to the non-time-resolved titration (Fig. 2(A)). This oligomer, however, possesses two classes of Eu3+ binding site as evidenced by the two lifetimes observed (Table 1) in H,O solution. The reason that only a single lifetime is observed in D20 is because the two Eu3+ species
"0
I
2
2
4
CEu~III)l/CDNA-PI
FIGURE 3. AmpUudes of the single (panel A) and double (panel B) exponential functions of the Eu3+ lumiaesce ncc decay curve (h, 579.20 nm, h, = 614 nm) as a function of equivalents of added Eu’+. A: pd(T)* (36 +I DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0); (Cl), 2993 1s. B: pd(C), (33 @l DNA-P in D,O, mM NaCl and 20 mM MES, pD 6.0); (0) 2850 ps; (A),761 ps.
LANTHANIDE ION PROBE8 OF DNA STRUC’I’URE.2. 199
present are in fast exchange relative to the ‘D,, excited-state lifetime ia that solvent [SJ. In fact, this is the only oligomer when? fast exchange between the “long” aud “short” lifm is obsemd. This fast exchange between “bug” and “short” lifetime env~~~ could be indicative of a simpler mechanism for Eu3+-induced oIigomers. A mechanism involving fewer aggregationiupd(T)sthaufortheother
intermedia@steps will occur more quickly than a complicated mechanism with many DISC steps. The time-resolved titration curve (Fig. 3(B)) of pd(C)s with Eu3+ resembles the time-resolved curves of the guanine-containing oligomers to a mter extent than those of pd(A), or pd(T),. Two classes of Eu3+ sites (2850 and 761 as) are present in oligo(dC),, with both sites possessing comparable afIinities for the Eu3+ ion. The long l&time titration curve (Fig 4) of pd(A)s is similar to the non-timeresolved titration curve for this oligomer with Eu’+. The long lifetime curve has the normal shape expected for a simple ~~b~~ between Eu3+ and a ligand until 1 @u3’]/@NA]. After this point, a tremendous increase in luminescence is observed. Thisincreaseinl~ nce may be the result of aggregation, which causes light
FIGuRe4. ~~~~~oe~~~of~~3+~~~y curves(a, 579.20 am, a, = 614 m) 88 a fmcticmof cquivakntsof addedEu3+. A: pd(A), (33 NMDNA-Pin DzO, 20 mMNaCiand20 mMME!&pD 6.0). (Cl) (~3+]/~NA-P]) < 1, 2313 cs; (El) (&u3*I,‘[DNA-PI)z 1, 2972 F; (A) (tau3+J/cDNA-PJ< 1, 578 w; (A) (@u3’]/&INA-P]>2: 1, 1025 p. B: The short lifetimetitrationcurve of Eu3+ tittmd iuto pi(A), showu on an eqmxled scab.
200
S. L, Klakamp and W. Dew. Horrocks, Jr.
scattering, and/or further binding of Eu3+ to the aggregates of pd(A)s. The short lifetime titration curve for pd(A)s is shown on an expanded scale in Fig. 4(B). This curve indicates that the Eu3+ species corresponding to the short lifetime class is decreasing in concentration up to one equivalent of added Et?+, and then it increases with the same shape as the long lifetime curve. The data of Figure 4 shows that upon Eu3+ addition to pd(A)s, two Eu3+ species are present. As Eu3+ is titrated into pd(A),, the short Eu3+ lifetime class is decreasing in coucentration while the long lifetime Eu3+ class is increasing in #n~n~on. This may be explained by an ~u~i~urn between two isomers, where one isomer is favored by increasing the concentration of Eu3+ ion. After 1 [Eu3+]/[DNA-P] two lifetimes different from those observed below one equivalent are measured, and these correspond to two Eu3+ bind~g sites, which are probably not ousting from the same isomeric equilibrium seen below one equivalent as evidenced by the different lifetimes. It appears that the two environments present below 1 equivalent are transformed, at into two sites which are probably not in an isomeric higher Eu3+ con~n~tions, ~u~ib~urn, or are at least in slow exchange with one another on the.time scales of the 5D0 excited-state lifetimes in D,O and H,O (Table 1). The time-resolved titration (Fig. 5(A)) of d-GMP by Eu3+ ion was performed in H,O for comparison with experiments on the other nucleotides, which were carried out in this solvent. The long and short lifetime classes follow the same general trends in behavior as the titration curves (in D,G) revealed in Figure 6 of the preceding paper [l]. Gnly one lifetime was observed in D,O for the Eu3+ complexes of d-CMP, d-AMP, and d-TMP, so in order to probe the characteristics of both Eu3+ binding environments, the titrations were carried out in H,O. The titration of d-TMP (Fig. 5(B)) with Eu3” ion indicates two classes of Eu3+ binding site. Even though the lifetime of Eu3+ aqua ion is 110 fis, the 110 I*Sclass is not assignable to this species. This can be proved by assuming the I10 @s class of Eu3+ ion is due to aqua Eu3+ ion. With this assumption, the 165 ps class titration curve can be analyzed for the dissociation constant &) of the 1:l Eu(d-TMP)+ complex [9]. When this is done, a K, of 76 PM is calculated. Using this K,,the theoretical 110 ps aqua Eu3 + curve can be plotted and compared with the shape of the experimental curve. The theoretical curve is concave-upward (Fig. 5(B)), whereas the experimental 110 ps curve is convex-upward, thus the experimental 110 ps curve cannot be due to aqua Eu3+ ion. The 110 p Eu3+ lifetime probably represents a class of site not resolvable experimentally from the 110 ps lifetime of the Eu3+ aqua ion. The 165 and 110 ps Eu 3+ binding site classes exhibit apparent similar afikities. Two classes of Eu3+ binding site are seen with d-AMP (Fig. S(C)). The shorter lifetime class of Eu3 + ion environment possesses a 115 ps lifetime similar to that of the 110 ps lifetime class in d-TMP. The 350 ~.lsclass of Eu3+ ion resembles the long lifetimes observed with d-GMP and d-CMP. The 350 +S class of Eu3+ site appears to coordinate more tightly than the 115 ks environment. Figure 6 shows the time-resolved titration curves of d-CMP with Eu3+. Two classes of Eu3+ binding site are observed. The 473 ps curve corresponds to a tight binding class of Eu3+ ion site relative to the 119 ps Eu3+ enviroument. De&&n&on
of the Number of Water Molecules Coordinated to E?achClass of Eu3+
The long and short lifetime Eu3+-containing species involve the coordination of the same number of H,O molecules (Table 1) as their respective guanosine analogues.
~NTHA~DE
I
ION PROBES
1
I
c
I
OF DNA
STRUCTURE.
2.
201
I I
. .
.
l
. . .
.
FIGURE 5. ~p~~~ of the two exponential functions of the Eu3+ luminescence decay curves (& 579.20 MI, )4,=614 nm) as a function of equivalents of Eiu’+ added to nucbotide monoplmsphates. A: d-GMp (58 glU in H,O, 20 mM NaCl and 20 mM MES, pH 6.0); (Cl) 374 cs; (A) 134 p. B: d-l%@ (139 pM inH,O, 20 &I NaCi and 20 mM MFiS, pH 6.0); (Cl) 165 ps; (A) 110 ps; (x) theomical 110 ps aqua Bu3+ curve (see text for explanation), C: d-AMP (104 pM in H,O, 20 mM NaCl end 20 mM MES, pH 6.0); (El) 350 gs; (A) 115 ps.
202
S. L. Klakamp and W. Dew. Horrocks, Jr.
EWIVALENTS
S.O
I
t
DF EuCIII) ADDED
t
1
,
B
a
FIGURE 6. Amplitudesof the two exponential Wctkms of the &I”’ lu~nw decay curves (X, 579.20 nm, &,, = 614 nm) 88 a function of equivalents of added Eu3+. A: d-CMP (214 CM in H,O, 20 n&l NaCl and 20 mh+iMES, pH 6.0); (Cl) 473 ps; 473 ps titration curve of d-CMP shown on an expanded scale.
(A)119 cs; B: The
The only possible exceptions are d-TMP and pd(T),. The nucleotide d-TMP has one Eu3+ ion class (165 p) that coordinates six H,O molecules instead of the usual two, while the 110 Jo class appears to bind nine water moleculea. Oligo(dT), deviates from the other &mers in possessing a long lifetime Eu’+ class of site which coordinates three Hz0 molecules instead of the usual two. Table 1 points out again that the tbymine-containing biomolecules differ fkom their counterparts made up of the uther three DNA bases. W Absorption Studies of pd(A),, pdo*,
pd(C&,, d-AMP, d-TMP, snd d-CMP
axon spectcel studies were performed to detemim if tme&4&ng occurs when Eu3+ is titrated into these molecules. Any major changes in the shapes of the absorption spectra would also be supportive of direct metal cootdinrttion of the base by Eu3+. The absoqtion spectrum (not shown) of pd(C)s exhibits a hyperchromic effect when one equivalent of Eu3+ is added. This suggests that pd(C)s is in a conformation wher7z the bases are at least partiaUy stacked prior to Eu3+ addition.
LANTHANIDE ION PROBES OF DNA STRUCTURE. 2.
203
Poly(dC) is known to form a double helix upon protonation of one-half of the bases [lo]. This double helix contains stacked C-C+ base pairs at close to neutral pH [ 111. The absorption spectrum (not shown) of pd(A)s indicates slight h~rch~~city upon Eu 3+ addition. Oligo(dT)s shows the least difkence between its absorption spectra (not shown) in the absence and pmsence of Bu3+ ion. None of the spectra of the oligomers indicate that base stacking is induced by the Eu3+ ion. However, evidence exists for Eu3+-induced base staking with the nuckotides. d-AMP exhibits hypochromicity in its absorption spectrum induced by the presence of Eu3+. The absorption spectra of d-TMP and d-CMP, however, show very little cation upon addition of Eu3+. DISCUSSION The oligomers studied hem all possess two classes of Eu3+ ion binding environments. This proves that two classes of Eu3+ binding site are not unique to guanine-containing oligomers. It was shown in the preceding paper [l J for the ~~on~g oligomers that the long lifetime component of Eu3+ huuinescence belongs to dimeric or polymeric species. Since the long lifetime components in pd(T),, pd(C)s and pd(A)s are similar to that of pd(G)s, it is assumed, by analogy, that the three oligomers examined here also form dimeric or polymeric structures. The coordinated water results of Table 1 suggest that the Eu3+ ion is coordinating to the phosphate oxygens, and possibly, by an outer sphere hydrogen bonding interaction, the unprotonated base nitrogen atoms of the 8-mers of adenine (N-l, N-7), guanine (N-7), and cytidine (N-3). If any in~~~on exists between Eu3+ and the base ~~gens, it must be of an outer sphere variety, since no change in the shape of the a~~~ spectra of the Eu3+-oligomer or -nu&otide complexes is seen. These oligomers exhibit a long lifetime component identical (within experimental error) with one another, and possess deprotonated base nitrogen atoms, a common denominator among pd(A)s, pd(C)s, and pd(G),. The N-7 nitrogen atom of pd(G)s and pd(A), (which is also deprotonated at N-l) in our proposed outer-sphere water bridging coordination of Eu3+ is important since N-7 is the only d~~o~t~ nitrogen atom in guanine at pH 6.0, while pd(C), (deprotonated at N-3) yields an identical lifetime in H,O. Oligo(d’I)s has only a protonated N-3 nitrogen at pH 6.0. A longer lifetime, 446 JJS, is also observed with pd(T)s, which is signikantly different from the long lifetimes of the other oligomers, indicating a sligbtly different environment for the Eu3+ ions bound to pd(T)s. Another possible explanation of the data in Table 1 is that the base nitrogens influence the nature of the aggregate formed between or among oligomers, which in turn bii Eu3+ solely through phosphate oxygens. Since pd(T)s has no deprotonated nitrogens, the structme of the aggregate may be different from those of the other oligomers. Perhaps this different structure controls the number of phosphate oxygens capable of coordinating the Eu3+ ion, thus allowing one more Hz0 molecule to coordinate the Eu3+ than in the other oligomers. The Eu3+ ions corresponding to the long lifetime class (in H,O) bind six or seven atoms from pd(A)s and pd(C)s most likely in an interstrand complex. The shorter lifetime components are consistent with sites involving Eu3+ ions binding to one or two asp oxygens of the DNA backbone of the oligomers. Base-stacking in the aggregates appears unlikely as evidenced by W absorption spectroscopy. The thymine nucleoti&, d-TMP, has a long lifetime (in H,O) significantly sborter than those of d-GMP, dCMP, and d-AMP. This observation lends further support to
244 S. L. Klakamp and W. L&W. Horrocks, Jr.
the idea that the base nitrogens influence the nature of the complex formed between Eu3+ and the oligomers and nucleotides containing guanine, cytidine, and adenine. The base nitrogens affect the Eu3+ coordination of the studied molecules most likely by participating in the Eu3+-induced aggregation process. Different polymeric str& tures conld place constraints on how many phosphate oxygens are able to coordiite a Eu3+ ion, yielding different Eu3’ excited-state lifetimes. The short lifetimes observed for the nucleotides suggest that this class of Eu3+ environment involves the ~~rdi~tion of only a single phosphate oxygen. The 110 hs lifetime of d-TMP implies that nine H,O water molecules are coordinated to Eu3+, the same number as in the Eu3+ aqua ion. The actual lifetime of the Eu3+-d-TMP complex is probably greater than 110 ps, but is experimentally unresolvable from that of the Eu3+ aqua ion. In summary, this research provides further evidence suggesting that the Eu3’ ion may coordinate the base nitrogens in an outer sphere mode in single-stranded DNA along with inner sphere coordination of the phosphate oxygens of the DNA backbone. The Eu3+ ion probably coordinates pd(T), and d-TMP solely through phosphate oxygens. The longer Eu3+ lifetime components in the nucl~ti&s, d-CMP and d-AMP, correspond to dimeric structures since these are identical (within experimental error) to the long lifetime observed in d-GMP, which was proved [I] to originate from a dimer. The 165 ps lifetime of Eu 3+ bound to d-TMP results either from the 1: 1 complex Eu(d-TMP)+ or from a dimer different in structure from those formed by the other oligomers. Although support for its assignment as a 1: 1 complex is provided by its similarity to the 191 us lifetime of the Eu(d-GMP)+ complex, it is difficult to understand how this species could be in slow exchange with the Eu3+ aqua ion. Table 1 indicates that two or three ligand atoms are bound to the 165 gs class of Eu3+ ion site in d-TMP, which could be easily explained by bidentate coordination of the Eu3+ by two phosphate oxygens. It was pointed out in the results section that the 110 ps lifetime is not simply attribntable to Eu3+ aqua ion, this species is obviously in fast exchange with the Eu3+ aqua ion and most likely involves the monodentate coronation of a single phosphate oxygen atom of d-TMP. The thymine base at pH 6.0 has only cat-bony1 ~~tio~ities capable of binding EL?+. It appears that carbonyl oxygens play no role in Eu3+ binding to DNA as further evidenced by the similarities of the observed longer lifetimes of pd(A), (which contains no carbonyl oxygen), pd(C), (which contains carbonyl oxygen, O-2), and pd(G), (which contains carbonyl oxygen, O-6). This trend also holds for the nucleotides. This work was supported by the National Science Foun~tion through grant No. CHEM-8821707. We thank Dr. R. C. Holz for helpfur dixussions and Drs. P. J. Breen and C. W. McNemar for writing some of the computer software used in the anaiysb of the data.
REFERENCES 1. S. L. Klakamp and W. Dew. Horrucks, Jr., J. Inorg. Eiochem. (1992) @receding paper iu this issue). 2. R. B. Martiu, Act. Chem. Res. 18, 32-38 (1985). 3. D. S. Gross and H. Sirnpki~BJ. Biol. Chem. 256, 9593-9598 (1981).
LANTHANIDE
ION PROBES OF DNA STRUCTURE. 2.
4. D. S. Gross, Ph.D.
205
Thesis, University of Colorado, Boulder, CO (1981). 5. M. P. Topal and J. R. Fresco, Biochemistry 19, 5531-5537 (1980). 6. D. Gersanovski, P. Colson, C. Houssier, and E. Fredericq, Biochem. Biophys. Actu 824, 313-323 (1985). 7. J. S. Fritz, R. T. Oliver, and K. J. Piehzyk, Anal. Chem. 30, 1111-1114 (1958). 8. W. Dew. Horrocks, Jr., V. K. Arkle, F. F. Liotta, and D. R. Sudnick, J. Am. Chem. sot. 105, 3455-3459 (1983). 9. C. W. McNemar and W. Dew. Horrocks, Jr., Anufyt. Biochem., 184, 35-38 (1990). 10. R. B. Innw, J. Mol. Biol. 9, 624-637 (1964). 11. D. M. Gray, T. Cui, and R. L. Ratliff, Nucleic Acids Res. 12, 7565-7580 (1984). Received October II, 1991; accepted December 4, 1991
of Chemistry,
The Pennsylvania
State
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ABSTRACT Oligo(dC),, oligo(dA),,
and oligo(dT& as well as d-CMP, d-AMP, and d-TMP, when complexed to ELI’+, possess two classes of Eu 3+ binding enviroument. The binding environments consist of two classes, tight sites which coordinate two Ha0 molecules, aud weaker sites which coordinate six or seven, analogous to the previously studied guanine-containing molecules. It is inferred that the tight class of Et?+ ion site observed with these oligomers and nucleotides corresponds to dimeric or polymeric structures. Comparison of the results for the guanine and ~-~~~ ~~~ ohgomers suggests that Eu3+ possibly coordinates base nitrogen atoms in the former and in an outer sphere mode (hydrogen bonding via the H,O molecules coordinated to Eu3+) in the species examined here.
In this paper the molecules oligo(dC),,
oligo(dA),, digo(dT),, d-CMP, d-AMP, and d-TMP are investigated by Eu3+ luminescence spectroscopy using the methods described in the preceding article [ 11. The major reason for undertakingthese studies was to determine if the base composition of DNA has any bearing on how Et?+ binds to single stranded DNA. It was suggested [l] that the N-7 ~~gens and/or O-6 oxygens of guanine could possibly coordinate to Eu3+. The base adenine has a N-7 nitrogen atom also, but no O-6 oxygen atom. The N-7 of adenine is also very weakly basic (pK, = - 1.6) [2], while the N-l of adenine is a much more basic site. The base cytosine has a carbonyl oxygen atom at O-2 and a nitrogen atom at N-3, while the base thymine possesses no unprotonated nitrogen atoms at pH 6.0. Transition metal ions favor coordination at N-3 of pyrimidine bases, and at the N-7
Present address, Division of Chemistry and Chemical Eng~~, California Institute of Technology, Pasadena, California. Address reprint requests and correspoudence to: W. D. Horrocks, Jr., Department of Chemistry, 152 Davey Lab, University park, PA 16802. Journal of Inorganic Biochemhtfy, 46, 193-205 (1992) 193 @ 1992EkevierSciencepublishingCo., Inc.. 655 Avenue of the Amnicas, NY, NY lOOtO Ol~~l~/~/$S.~
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S. L. Klakamp and W. DeW. Horrocks, Jr.
and N-l sites of purine bases [2]. Controversy exists in the literature [3-61 over whether Tb3+, or by analogy, Eu3+, coordinates-to the N-7 of guanine residues in addition to the commonly accepted phosphate oxygen coordination. Our study of the B-mers of the four DNA bases was carried out in order to provide insight into the role of the base in Ln3+ bi~ng. axon of the nucl~des allows a closer inspection of the monomeric units of which the 8-mers consist, and provides data which suggest that the Eu3 + complexes of d-CMP and d-AMP form dimeric structures in solution. These are the first studies to look directly at Ln3+ ion coordination to non-guaninecontaining DNA. MATERIALS AND METHODS Materials The o~g~~x~ucl~ti&s were purchased from Pharmacia, Inc. and used as supplied. All the nucleotides studied were purchased from Sigma Chemical Co. and used without further purification. Concentrations were determined spectrophotomet&ally for the studied biomolecules (with lot numbers in parentheses) using the following molar absorptivities at 260 nm: pd(C), (00815-114), 7200; pd(Q (~107~), 7800; pd(A)s (~35) 12,400; d-CMP (128C-7000), 7500; d-AMP (128C-7030), 15,400; d-TMP (63f-7000), 9200. All oligomers and nucleotides were dissolved in 20 mM NaCl and 20 mM MES buffer (PH 6.0) made up in doubly distilled H,O or D,O (Aldrich Chemical Co., 99.8%). EuCl, * 6H,O and ThCl * 6H,O were obtained from Aldrich Chemical Co. and used as supplied. Eu 3, solutions were standard&d by a chelometric method 173. Eu3 + Time-Resolved Luminescence Titrations The details of the laser experiments are identical to those described in the preceding paper [ 1J. Luminescence decays at each point in a titration were collected for 15 min (9000 transients) and corrected for fluctuations in laser power during the course of an experiment, The titrations of the oligomers were performed in D,O, and the time-resolved titrations of the nucleotides were done in H,O. Luminescence decays were analyzed as described in the preceding article [l]. The lifetimes of d-TMP, d-AMP, d-CMP, d-GMP, pd(C&,, and pd(T)s were held constant in the Marquardt analysis of their respective fits. The lifetimes determined for the pd(A)s system changed sig~fi~~y after one equivalent of Eu3+ ion was added, and therefore two sets of lifetime values, one set before and one set after the addition of one equivalent, were held constant in the nonlinear regression analysis (as described in the legend to Fig. 4). In the Marquardt analysis of d-TMP, one lifetime component was held constant at 110 ps. When all parameters were allowed to ride freely in the nonlinear regression analysis for d-TMP, no two lifetime components values were obtained consistently from a double exponential fit. Upon holding one lifetime component constant at 110 ps in the Marquardt analysis, a very consistent 165 ps lifetime value was generated, as shown by the standard deviation for this component in Table 1. The lifetime value of 110 ps was a logical guess (not arrived at by an average of lifetime values as in the other systems) for the second lifetime component, since single exponential analyses of the luminescence decay curves for d-TMP gave a lifetime value of 119 ps with very poor fits to the decay curves. The non-time-resolved titrations with Eu3+ were performed identically to those described previously [ 11.
L A N T H A N I D E ION PROBES O F D N A S T R U C T U R E . 2.
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TABLE 1. Determination of the Number of Water Molecules, q, Coordinated to EU3+ Bound to non-guanine-containing Oligomers and Nucleotides Oligomer pd(A)sc [Eu3+ ] < [DNA-P] [Eu3+ ] ~e [DNA-P] pd(T)sc pd(C)sc d_AMPc d_CMPc
"H 20(/~s) a
• D20(/zs)a
qb
311 + 10 136 + 5 311 5:10 136 + 5 446 5:35 143 5:6 333 5:26 155 5:17 350 5:16 115+4 473 5:95
578 + 41 2313 5:90 1025 + 62 2972 + 37 2993 + 76
2.0 + 0.5 7.3 + 0.5 2.1 + 0.5 7.4 5:0.5 2.8 5:0.5 6.4 + 0.5 1.8 + 0.5 6.4 + 0.5 2.7 + 0.5 8.8+0.5 1.9 + 0.5 8 . 5 + 0.5 6.0 5:0.5 9 . 2 + 0.5
761 5:104 2850 5:43 3198 + 118 3038 5:38
119 + 3
d_TMPc
165 5:16
2972 5:22
110 'q
a Errors shown are the standard deviation associated with each lifetime measurement. b Errors are ~ t e d as in D. Skoog and D. West, Fundamentals of Analytical Chemistry, Saunders College Publishing, Philadelphia, 1982, Chapter 3, pp. 39-82. c Eu3+ oligomer and nucleotide concentrations are those employed in their respective time-resolved titrations (Figs.3-6). dNo standard deviation is assigned because 110 ~ts is an estimate.
RESULTS N o n - T i m e - R e s o l v e d T i t r a t i o n s o f p d ( A ) e, p d ( C ) 8, a n d p d ( T ) s The excitation spectra o f the oligomers and nucleotides studied here are shown in Figure 1. The spectra o f the oligomers are very broad and similar to those o f the guanine-containing oligomers. In the non-time-resolved and time-resolved titrations an excitation wavelength o f 579.20 nm was used to eliminate, as much as possible, the contribution o f Eu 3+ aqua ion luminescence to the observed signal. The excitation peaks for the nucleotides are narrower than those o f the oligomers, consistent with the smaller number o f Eu 3 + environments capable o f being adopted by the nucleotide complex. A s seen in Figure 2, the curve for pd(T) 8 titrated with Eu 3+ breaks at 0.35 [ E u 3 + ] / [ D N A - P ] . The increase in luminescence beyond this point is largely due to that o f the aqua Eu 3 + ion. It appears that three phosphate moieties are coordinating per Eu 3+ in o l i g o ( d T ) s - - t h e number that would be expected based on charge neutralization considerations. However, evidence exists, which will be discussed later, for a possible intermolecular interaction (aggregate) between or among Eu3+-pd(T)s oligomers. The extent o f aggregation seen with lxl(T) s is, however, less than that with the guanine-containing oligumers. In any case, Eu 3+ binds PriG')8 strongly until 0.35 equivalents o f Eu 3+ have been added, which corresponds to the charge neutralization point. This is the only 8-mer investigated which exhibits this expected type o f behavior. The non-time-resolved titration (Fig. 2(13)) o f pd(C)s with Eu 3+ indicates behavior shnilar to that seen in the titration o f pal(G) 8 with Eu 3+ (Fig. 2 in preceding paper). The titration curve for lxi(C) 8 shows that Eu 3+ is bound tightly to pd(C) s until - 0 . 5 to 1.0 [ E u 3 + ] / [ D N A - P ] . The similarity seen between the pd(C) 8 and 1x1(O)8 titrations lies in the fact that both curves continue to indicate Eu 3+ binding even up
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S. L. KIakamp and W. De W. Homocks,Jr,
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FIGURE 1. 7FO-+‘D, excitation spectra (h
= 614 mu) of Eu3+ bound to various nucleotides and oligonucleotides. (Top panel): A: oligo(dC), (26 PM DNA-P); B: oligo(dA)8 (33 FM DNA-P); C: oBgo(dT)~ (27 pM DNA-P) at 1.0 ~~+]/[DNA-P]. (Bottom panel): A: d-TMP (35 HM); B: d-AMP (38 CM); C: d-CMP (33 FM) at 1.0 @h3+]/DNA-P].
to five added equivalents of Eu 3+. The slope of the line produced by the simple
addition of Eu3+ to buffer is less than the slope of the pd(C), titration curve from one to five equivalents. The increase in luminescence from one to five [Eu”+]/ [DNA-P] rest&s from the following three factors: 1) Eu3+ aqua ion luminescence, 2) additional biing of Eu3+ to pd(C), (single-stranded or aggregate), and (3) aggregation (crosslinking) of pd(C)s oligomers via Fu3+ ions bound to pd(C)s. In this last case, Eu3+ luminescence would increase owing to the dehydration of the inner coordination sphere of Eu3 + ions by eliminating a deexcitation pathway. nl~~ also in Fig. 2(C) is the non-~-~lv~ ti~ti~ of pd(A), with Eu3+. This curve is strikingly different from the curves for the other El-mers. Eu3+ ion coordinates pd(A)s tightly until [Eu3+],‘[DNA-P] = 1.0. The sharp increase in luminescence observed after one equivalent of Eu3+ may be explained by the more pronounced ability of pd(A)s to aggregate at lower concentrations of Eu3+. This is
LANTHANIDE
100 A
I
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FIGURE 2. Non-time-resolved titration with Eu3+ lumiuescence ()Lx = 579.20 um, &,,, = 614 run) plotted as a function of added Eu3+. A: pd(‘l& (0) (31 pM DNA-P in D,O, 20 mh4 NaCl and 20 mM MES, pD 6.0). B: pd(C), (0) (33 pM DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0). C: pd(A), (0) (31 CM DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0). (A) the simple addition of Eu3+ into buffer.
198
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supported by the turbidity observed for some pd(A)s solutions which was detected visually after the addition of - 4 to 5 equivalents of Eu3+. Additional data suggesting this stronger tendency towards aggregation is provided by an (A32O/A260) ratio of 0.18 at 3.0 [Eu]/[DNA-P] for pd(A), versus values of this ratio of 0.009 and 0.096 for pd(T), and pd(C),, respectively. Time-Resolved Eu3 + Luminescence Titration Studies The time-resolved titration of pdQ, with Eu3+ in D,O (Fig. 3) consists of a single exponential and, not surprisingly, corresponds closely to the non-time-resolved titration (Fig. 2(A)). This oligomer, however, possesses two classes of Eu3+ binding site as evidenced by the two lifetimes observed (Table 1) in H,O solution. The reason that only a single lifetime is observed in D20 is because the two Eu3+ species
"0
I
2
2
4
CEu~III)l/CDNA-PI
FIGURE 3. AmpUudes of the single (panel A) and double (panel B) exponential functions of the Eu3+ lumiaesce ncc decay curve (h, 579.20 nm, h, = 614 nm) as a function of equivalents of added Eu’+. A: pd(T)* (36 +I DNA-P in D,O, 20 mM NaCl and 20 mM MES, pD 6.0); (Cl), 2993 1s. B: pd(C), (33 @l DNA-P in D,O, mM NaCl and 20 mM MES, pD 6.0); (0) 2850 ps; (A),761 ps.
LANTHANIDE ION PROBE8 OF DNA STRUC’I’URE.2. 199
present are in fast exchange relative to the ‘D,, excited-state lifetime ia that solvent [SJ. In fact, this is the only oligomer when? fast exchange between the “long” aud “short” lifm is obsemd. This fast exchange between “bug” and “short” lifetime env~~~ could be indicative of a simpler mechanism for Eu3+-induced oIigomers. A mechanism involving fewer aggregationiupd(T)sthaufortheother
intermedia@steps will occur more quickly than a complicated mechanism with many DISC steps. The time-resolved titration curve (Fig. 3(B)) of pd(C)s with Eu3+ resembles the time-resolved curves of the guanine-containing oligomers to a mter extent than those of pd(A), or pd(T),. Two classes of Eu3+ sites (2850 and 761 as) are present in oligo(dC),, with both sites possessing comparable afIinities for the Eu3+ ion. The long l&time titration curve (Fig 4) of pd(A)s is similar to the non-timeresolved titration curve for this oligomer with Eu’+. The long lifetime curve has the normal shape expected for a simple ~~b~~ between Eu3+ and a ligand until 1 @u3’]/@NA]. After this point, a tremendous increase in luminescence is observed. Thisincreaseinl~ nce may be the result of aggregation, which causes light
FIGuRe4. ~~~~~oe~~~of~~3+~~~y curves(a, 579.20 am, a, = 614 m) 88 a fmcticmof cquivakntsof addedEu3+. A: pd(A), (33 NMDNA-Pin DzO, 20 mMNaCiand20 mMME!&pD 6.0). (Cl) (~3+]/~NA-P]) < 1, 2313 cs; (El) (&u3*I,‘[DNA-PI)z 1, 2972 F; (A) (tau3+J/cDNA-PJ< 1, 578 w; (A) (@u3’]/&INA-P]>2: 1, 1025 p. B: The short lifetimetitrationcurve of Eu3+ tittmd iuto pi(A), showu on an eqmxled scab.
200
S. L, Klakamp and W. Dew. Horrocks, Jr.
scattering, and/or further binding of Eu3+ to the aggregates of pd(A)s. The short lifetime titration curve for pd(A)s is shown on an expanded scale in Fig. 4(B). This curve indicates that the Eu3+ species corresponding to the short lifetime class is decreasing in concentration up to one equivalent of added Et?+, and then it increases with the same shape as the long lifetime curve. The data of Figure 4 shows that upon Eu3+ addition to pd(A)s, two Eu3+ species are present. As Eu3+ is titrated into pd(A),, the short Eu3+ lifetime class is decreasing in coucentration while the long lifetime Eu3+ class is increasing in #n~n~on. This may be explained by an ~u~i~urn between two isomers, where one isomer is favored by increasing the concentration of Eu3+ ion. After 1 [Eu3+]/[DNA-P] two lifetimes different from those observed below one equivalent are measured, and these correspond to two Eu3+ bind~g sites, which are probably not ousting from the same isomeric equilibrium seen below one equivalent as evidenced by the different lifetimes. It appears that the two environments present below 1 equivalent are transformed, at into two sites which are probably not in an isomeric higher Eu3+ con~n~tions, ~u~ib~urn, or are at least in slow exchange with one another on the.time scales of the 5D0 excited-state lifetimes in D,O and H,O (Table 1). The time-resolved titration (Fig. 5(A)) of d-GMP by Eu3+ ion was performed in H,O for comparison with experiments on the other nucleotides, which were carried out in this solvent. The long and short lifetime classes follow the same general trends in behavior as the titration curves (in D,G) revealed in Figure 6 of the preceding paper [l]. Gnly one lifetime was observed in D,O for the Eu3+ complexes of d-CMP, d-AMP, and d-TMP, so in order to probe the characteristics of both Eu3+ binding environments, the titrations were carried out in H,O. The titration of d-TMP (Fig. 5(B)) with Eu3” ion indicates two classes of Eu3+ binding site. Even though the lifetime of Eu3+ aqua ion is 110 fis, the 110 I*Sclass is not assignable to this species. This can be proved by assuming the I10 @s class of Eu3+ ion is due to aqua Eu3+ ion. With this assumption, the 165 ps class titration curve can be analyzed for the dissociation constant &) of the 1:l Eu(d-TMP)+ complex [9]. When this is done, a K, of 76 PM is calculated. Using this K,,the theoretical 110 ps aqua Eu3 + curve can be plotted and compared with the shape of the experimental curve. The theoretical curve is concave-upward (Fig. 5(B)), whereas the experimental 110 ps curve is convex-upward, thus the experimental 110 ps curve cannot be due to aqua Eu3+ ion. The 110 p Eu3+ lifetime probably represents a class of site not resolvable experimentally from the 110 ps lifetime of the Eu3+ aqua ion. The 165 and 110 ps Eu 3+ binding site classes exhibit apparent similar afikities. Two classes of Eu3+ binding site are seen with d-AMP (Fig. S(C)). The shorter lifetime class of Eu3 + ion environment possesses a 115 ps lifetime similar to that of the 110 ps lifetime class in d-TMP. The 350 ~.lsclass of Eu3+ ion resembles the long lifetimes observed with d-GMP and d-CMP. The 350 +S class of Eu3+ site appears to coordinate more tightly than the 115 ks environment. Figure 6 shows the time-resolved titration curves of d-CMP with Eu3+. Two classes of Eu3+ binding site are observed. The 473 ps curve corresponds to a tight binding class of Eu3+ ion site relative to the 119 ps Eu3+ enviroument. De&&n&on
of the Number of Water Molecules Coordinated to E?achClass of Eu3+
The long and short lifetime Eu3+-containing species involve the coordination of the same number of H,O molecules (Table 1) as their respective guanosine analogues.
~NTHA~DE
I
ION PROBES
1
I
c
I
OF DNA
STRUCTURE.
2.
201
I I
. .
.
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. . .
.
FIGURE 5. ~p~~~ of the two exponential functions of the Eu3+ luminescence decay curves (& 579.20 MI, )4,=614 nm) as a function of equivalents of Eiu’+ added to nucbotide monoplmsphates. A: d-GMp (58 glU in H,O, 20 mM NaCl and 20 mM MES, pH 6.0); (Cl) 374 cs; (A) 134 p. B: d-l%@ (139 pM inH,O, 20 &I NaCi and 20 mM MFiS, pH 6.0); (Cl) 165 ps; (A) 110 ps; (x) theomical 110 ps aqua Bu3+ curve (see text for explanation), C: d-AMP (104 pM in H,O, 20 mM NaCl end 20 mM MES, pH 6.0); (El) 350 gs; (A) 115 ps.
202
S. L. Klakamp and W. Dew. Horrocks, Jr.
EWIVALENTS
S.O
I
t
DF EuCIII) ADDED
t
1
,
B
a
FIGURE 6. Amplitudesof the two exponential Wctkms of the &I”’ lu~nw decay curves (X, 579.20 nm, &,, = 614 nm) 88 a function of equivalents of added Eu3+. A: d-CMP (214 CM in H,O, 20 n&l NaCl and 20 mh+iMES, pH 6.0); (Cl) 473 ps; 473 ps titration curve of d-CMP shown on an expanded scale.
(A)119 cs; B: The
The only possible exceptions are d-TMP and pd(T),. The nucleotide d-TMP has one Eu3+ ion class (165 p) that coordinates six H,O molecules instead of the usual two, while the 110 Jo class appears to bind nine water moleculea. Oligo(dT), deviates from the other &mers in possessing a long lifetime Eu’+ class of site which coordinates three Hz0 molecules instead of the usual two. Table 1 points out again that the tbymine-containing biomolecules differ fkom their counterparts made up of the uther three DNA bases. W Absorption Studies of pd(A),, pdo*,
pd(C&,, d-AMP, d-TMP, snd d-CMP
axon spectcel studies were performed to detemim if tme&4&ng occurs when Eu3+ is titrated into these molecules. Any major changes in the shapes of the absorption spectra would also be supportive of direct metal cootdinrttion of the base by Eu3+. The absoqtion spectrum (not shown) of pd(C)s exhibits a hyperchromic effect when one equivalent of Eu3+ is added. This suggests that pd(C)s is in a conformation wher7z the bases are at least partiaUy stacked prior to Eu3+ addition.
LANTHANIDE ION PROBES OF DNA STRUCTURE. 2.
203
Poly(dC) is known to form a double helix upon protonation of one-half of the bases [lo]. This double helix contains stacked C-C+ base pairs at close to neutral pH [ 111. The absorption spectrum (not shown) of pd(A)s indicates slight h~rch~~city upon Eu 3+ addition. Oligo(dT)s shows the least difkence between its absorption spectra (not shown) in the absence and pmsence of Bu3+ ion. None of the spectra of the oligomers indicate that base stacking is induced by the Eu3+ ion. However, evidence exists for Eu3+-induced base staking with the nuckotides. d-AMP exhibits hypochromicity in its absorption spectrum induced by the presence of Eu3+. The absorption spectra of d-TMP and d-CMP, however, show very little cation upon addition of Eu3+. DISCUSSION The oligomers studied hem all possess two classes of Eu3+ ion binding environments. This proves that two classes of Eu3+ binding site are not unique to guanine-containing oligomers. It was shown in the preceding paper [l J for the ~~on~g oligomers that the long lifetime component of Eu3+ huuinescence belongs to dimeric or polymeric species. Since the long lifetime components in pd(T),, pd(C)s and pd(A)s are similar to that of pd(G)s, it is assumed, by analogy, that the three oligomers examined here also form dimeric or polymeric structures. The coordinated water results of Table 1 suggest that the Eu3+ ion is coordinating to the phosphate oxygens, and possibly, by an outer sphere hydrogen bonding interaction, the unprotonated base nitrogen atoms of the 8-mers of adenine (N-l, N-7), guanine (N-7), and cytidine (N-3). If any in~~~on exists between Eu3+ and the base ~~gens, it must be of an outer sphere variety, since no change in the shape of the a~~~ spectra of the Eu3+-oligomer or -nu&otide complexes is seen. These oligomers exhibit a long lifetime component identical (within experimental error) with one another, and possess deprotonated base nitrogen atoms, a common denominator among pd(A)s, pd(C)s, and pd(G),. The N-7 nitrogen atom of pd(G)s and pd(A), (which is also deprotonated at N-l) in our proposed outer-sphere water bridging coordination of Eu3+ is important since N-7 is the only d~~o~t~ nitrogen atom in guanine at pH 6.0, while pd(C), (deprotonated at N-3) yields an identical lifetime in H,O. Oligo(d’I)s has only a protonated N-3 nitrogen at pH 6.0. A longer lifetime, 446 JJS, is also observed with pd(T)s, which is signikantly different from the long lifetimes of the other oligomers, indicating a sligbtly different environment for the Eu3+ ions bound to pd(T)s. Another possible explanation of the data in Table 1 is that the base nitrogens influence the nature of the aggregate formed between or among oligomers, which in turn bii Eu3+ solely through phosphate oxygens. Since pd(T)s has no deprotonated nitrogens, the structme of the aggregate may be different from those of the other oligomers. Perhaps this different structure controls the number of phosphate oxygens capable of coordinating the Eu3+ ion, thus allowing one more Hz0 molecule to coordinate the Eu3+ than in the other oligomers. The Eu3+ ions corresponding to the long lifetime class (in H,O) bind six or seven atoms from pd(A)s and pd(C)s most likely in an interstrand complex. The shorter lifetime components are consistent with sites involving Eu3+ ions binding to one or two asp oxygens of the DNA backbone of the oligomers. Base-stacking in the aggregates appears unlikely as evidenced by W absorption spectroscopy. The thymine nucleoti&, d-TMP, has a long lifetime (in H,O) significantly sborter than those of d-GMP, dCMP, and d-AMP. This observation lends further support to
244 S. L. Klakamp and W. L&W. Horrocks, Jr.
the idea that the base nitrogens influence the nature of the complex formed between Eu3+ and the oligomers and nucleotides containing guanine, cytidine, and adenine. The base nitrogens affect the Eu3+ coordination of the studied molecules most likely by participating in the Eu3+-induced aggregation process. Different polymeric str& tures conld place constraints on how many phosphate oxygens are able to coordiite a Eu3+ ion, yielding different Eu3’ excited-state lifetimes. The short lifetimes observed for the nucleotides suggest that this class of Eu3+ environment involves the ~~rdi~tion of only a single phosphate oxygen. The 110 hs lifetime of d-TMP implies that nine H,O water molecules are coordinated to Eu3+, the same number as in the Eu3+ aqua ion. The actual lifetime of the Eu3+-d-TMP complex is probably greater than 110 ps, but is experimentally unresolvable from that of the Eu3+ aqua ion. In summary, this research provides further evidence suggesting that the Eu3’ ion may coordinate the base nitrogens in an outer sphere mode in single-stranded DNA along with inner sphere coordination of the phosphate oxygens of the DNA backbone. The Eu3+ ion probably coordinates pd(T), and d-TMP solely through phosphate oxygens. The longer Eu3+ lifetime components in the nucl~ti&s, d-CMP and d-AMP, correspond to dimeric structures since these are identical (within experimental error) to the long lifetime observed in d-GMP, which was proved [I] to originate from a dimer. The 165 ps lifetime of Eu 3+ bound to d-TMP results either from the 1: 1 complex Eu(d-TMP)+ or from a dimer different in structure from those formed by the other oligomers. Although support for its assignment as a 1: 1 complex is provided by its similarity to the 191 us lifetime of the Eu(d-GMP)+ complex, it is difficult to understand how this species could be in slow exchange with the Eu3+ aqua ion. Table 1 indicates that two or three ligand atoms are bound to the 165 gs class of Eu3+ ion site in d-TMP, which could be easily explained by bidentate coordination of the Eu3+ by two phosphate oxygens. It was pointed out in the results section that the 110 ps lifetime is not simply attribntable to Eu3+ aqua ion, this species is obviously in fast exchange with the Eu3+ aqua ion and most likely involves the monodentate coronation of a single phosphate oxygen atom of d-TMP. The thymine base at pH 6.0 has only cat-bony1 ~~tio~ities capable of binding EL?+. It appears that carbonyl oxygens play no role in Eu3+ binding to DNA as further evidenced by the similarities of the observed longer lifetimes of pd(A), (which contains no carbonyl oxygen), pd(C), (which contains carbonyl oxygen, O-2), and pd(G), (which contains carbonyl oxygen, O-6). This trend also holds for the nucleotides. This work was supported by the National Science Foun~tion through grant No. CHEM-8821707. We thank Dr. R. C. Holz for helpfur dixussions and Drs. P. J. Breen and C. W. McNemar for writing some of the computer software used in the anaiysb of the data.
REFERENCES 1. S. L. Klakamp and W. Dew. Horrucks, Jr., J. Inorg. Eiochem. (1992) @receding paper iu this issue). 2. R. B. Martiu, Act. Chem. Res. 18, 32-38 (1985). 3. D. S. Gross and H. Sirnpki~BJ. Biol. Chem. 256, 9593-9598 (1981).
LANTHANIDE
ION PROBES OF DNA STRUCTURE. 2.
4. D. S. Gross, Ph.D.
205
Thesis, University of Colorado, Boulder, CO (1981). 5. M. P. Topal and J. R. Fresco, Biochemistry 19, 5531-5537 (1980). 6. D. Gersanovski, P. Colson, C. Houssier, and E. Fredericq, Biochem. Biophys. Actu 824, 313-323 (1985). 7. J. S. Fritz, R. T. Oliver, and K. J. Piehzyk, Anal. Chem. 30, 1111-1114 (1958). 8. W. Dew. Horrocks, Jr., V. K. Arkle, F. F. Liotta, and D. R. Sudnick, J. Am. Chem. sot. 105, 3455-3459 (1983). 9. C. W. McNemar and W. Dew. Horrocks, Jr., Anufyt. Biochem., 184, 35-38 (1990). 10. R. B. Innw, J. Mol. Biol. 9, 624-637 (1964). 11. D. M. Gray, T. Cui, and R. L. Ratliff, Nucleic Acids Res. 12, 7565-7580 (1984). Received October II, 1991; accepted December 4, 1991