Thermodynamics of DNA containing very stable chemically modified base pairs

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymer DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T(m)) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T(m) independent of DNA length and equal to T(infinity), the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2 omega-a)S/(1-S) and S = exp[DeltaS(T(infinity)-T)/(RT)] where omega is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and DeltaS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for omega=1, and n<0.1N for omega>1, where N is the number of ordinary base pairs in the DNA chain.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Estimation of the diversity between DNA calorimetric profiles,differential melting curves and corresponding melting temperatures

The Poland–Fixman–Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (H_GC?H_AT) between the helix‐coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with H_GC?H_AT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed H_GC?H_AT, the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (T_cal?T_m) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (T_cal?T_m) enlarge with the temperature melting range of the helix‐coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Evidence for long-range interactions in DNA. Analysis of melting curves of block polymers d(C15A15)·d(T15G15), d(C20A15)·d(T15G20), and d(C20A10)·d(T10G20)

The influence of one DNA region on the stability of an adjoining region (telestability) was examined. Melting curves of three block DNA's, d(C₁₅A₁₅)·d(T₁₅G₁₅), d(C₂₀A₁₅)·d(T₁₅G₂₀), and d(C₂₀A₁₀)·d(T₁₀G₂₀) were analyzed in terms of the nearest neighbor Ising model. Comparisons of predicted and experimental curves were made in 0.01 M and 0.1 M sodium ion solutions. The nearest neighbor formalism was also employed to analyze block DNA transition in the presence of actinomycin, a G·C specific molecule. The results show that nearest neighbor base-pair interaction cannot predict the melting curves of the block DNA's. Adjustments in theoretical parameters to account for phosphate repulsion assuming a B conformation throughout the DNA's do not alter this conclusion. Changes in the theoretical parameters, which provide good overall agreement, are consistent with a substantial stabilization of the A·T region nearest the G·C block. The melting temperature T A·T for the average A·T pari in d(C₂₀A₁₀)·d(T₁₀G₂₀), with 10 A·T pairs, appears to be 4°C greater than T_A·T for d(C₁₅A₁₅)·d(T₁₅G₁₅) and d(C₂₀A₁₅)·d(T₁₅G₂₀), both with 15 A·T pairs. Actinomycin bound to the G·C end effectively stabilizes the A·T end by 9°C. These results indicate a long-range contribution to the interactions governing DNA stability. A possible mechanism for these interactions will be discussed.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

A circular dichroism study of the primary structures of three crab satellite DNA's rich in A:T base pairs

We have measured the circular dichroism spectra and have obtained melting curves for samples of purified satellite DNA's from three species of crab, Cancer borealis, Gecarcinus lateralis, and Callinectes sapidus. Although all three satellite DNA's have the same density in neutral CsCl, their circular dichroism spectra and melting and reassociation characteristics are different, as are other properties of these DNA's. The circular dichroism spectra are analyzed to give estimates of nearest-neighbor frequencies. We estimate that about 90% of the Cancer borealis satellite DNA and over 80% of the Gecarcinus lateralis satellite DNA consist of d(AT)_n:d(AT)_n sequences, while the serially repeating unit of the Callinectes sapidus satellite DNA may not contain any extended d(AT)_n:d(AT)_n sequences. The results from the circular dichroism spectra for the Cancer borealis, Gecarcinus lateralis, and the Callinectes sapidus satellite DNA's are in agreement with base composition analyses, which give 2.4, 8, and 25% G+C, respectively, for these DNA's.     

Nuclear and Kinetoplastic DNA from Trypanosomes*

SYNOPSIS. Data concerning the DNA and RNA content of 3 Trypanosoma species (T. equiperdum, T. gambiense and T. cruzi) are given. Kinetoplastic DNA was fractionated and separated from nuclear DNA by ultracentrifugation in a C_S2SO₄ gradient after complexation by mercury or silver ions. Buoyant densities in the analytic ultracentrifuge, base composition and melting point of these DNA's were studied.     

Synthesis of 2H,13C-Labelled 2′-Deoxynucleosides and Their Site Specific Incorporation into Oligo-DNA for Structural Studies via Relaxation Time Measurements

Abstract

We have recently shown¹ the usefulness of ²H, ¹³C-labelled 2′-deoxynu-cleoside building blocks for structural studies via relaxation time measurements. The synthesis of phosphoramidite blocks 11 and 12 for their site-specific incorporation (indicated by underlines) into the d⁵′(¹C²G³ A ⁴ T ⁵ T ⁶ A ⁷ A ⁸ T ⁹C¹⁰G)₂ ³′ is briefly described for studying the T₁ and T_1[sgrave] relaxations of ²H and ¹³C at specific deuterated carbons in a large molecule.     

9-(6-Deoxy- β -D-Allofuranosyl)Adenine Cyclic 3′,5′ -Phosphor-Amidate: A New Cyclic AMP Amide Derivative Containing an Equatorial Methyl Group at the 5′-Position

Abstract

R _P and S _P phosphorus diastereoisomers of the title compound (2) are prepared from the corresponding cyclic monophosphate. Solution conformation of the dioxaphosphor-inane ring and hydrolysis of R _p-2 and S _p-2 are studied and compared with those of the phosphorus diastereoisomers of the isomeric compound that contains the 5′-methyl group in the axial position.     

Synthesis of (R - and (S)-1-[[2-Hydroxy-1-(aminomethyl)ethoxy]methyl]-5- benzyluracil,Potent Inhibitors of Uridine Phosphorylase

Abstract

Optically pure (R)- and (S)-1-[[2-hydroxy-1-(aminomethyl) ethoxy]methyl]-5-benzyluracil [(R)-AHPBU and (S)-AHPBU, respectively], two potent uridine phosphorylase inhibitors, have been synthesized via multi-step syntheses starting from independent chiral compounds. The activity of (R)-AHPBU, (S)-AHPBU, and (R,S-AHPBU which have been previously synthesized, on the inhibition of uridine phosphorylase from Sarcoma-180 cells has been studied and compared. The K. values for (R,S)-, (R)- and (S)-AHPBU were determined to be 15·2.3, 17·2.7 and 16·2.0 nM, respectively. This indicates that (R) and (S) optical enantiomers have the same affinity for binding to uridine phosphorylase. These acyclic pyrimidine amino nucleoside analogues represent a new class of potent uridine phosphorylase inhibitors, which has a bulky hydrophobic substituent at the 5-position in the uracil base, yet has remarkably high water solubility.     

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T₄ single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ～20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (～ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of f_T(T), the fraction of triplex as a function of temperature, was obtained. Analysis of f_T vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔH_T = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔS_T = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from f_T = 0 to 0.55 and a higher, sharper transition above f_T = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′_T = 44.8 ± 9.6 kcal/mol and ΔS′_T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′_T = 128.0 ± 2.3 kcal/mol and ΔS′_T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.     

Structure of 2′,5′-Linked Tetraribonucleotide Loops: A Novel RNA Motif

Abstract

We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U_2′p5′U_2′p5′C_2′p5′G_2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.     

31P-15N One-Bond Couplings of Diastereoisomeric Adenosine Cyclic 3′,5′-Phosphoramidates

Abstract

¹J(³¹P¹⁵N) coupling constants of R _p and S _p adeno-sine cyclic 3′,5′-phosphoramidates (1), -N-methylphosphor-amidates (2) and -N,N-dimethylphosphoramidates (3) increase in the order of 1<2<3 and obey the Stec rule (J(R _p)< J(S _p)). A possible interpretation of coupling constant differences based on differences in substituent electronegativities and variation in hybridization at nitrogen atom, is suggested.     

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G–A cross-linking PBD–duocarmycin dimers

The pyrrolobenzodiazepine (PBD) and duocarmycin families are DNA-interactive agents that covalently bond to guanine (G) and adenine (A) bases, respectively, and that have been joined together to create synthetic dimers capable of cross-linking G–G, A–A, and G–A bases. Three G–A alkylating dimers have been reported in publications to date, with defined DNA-binding sites proposed for two of them. In this study we have used molecular dynamics simulations to elucidate preferred DNA-binding sites for the three published molecular types. For the PBD–CPI dimer UTA-6026 (1), our simulations correctly predicted its favoured binding site (i.e., 5′-C(G)AATTA-3′) as identified by DNA cleavage studies. However, for the PBD–CI molecule (‘Compound 11’, 3), we were unable to reconcile the results of our simulations with the reported preferred cross-linking sequence (5′-ATTTTCC(G)-3′). We found that the molecule is too short to span the five base pairs between the A and G bases as claimed, but should target instead a sequence such as 5′-ATTTC(G)-3′ with two less base pairs between the reacting G and A residues. Our simulation results for this hybrid dimer are also in accord with the very low interstrand cross-linking and in vitro cytotoxicity activities reported for it. Although a preferred cross-linking sequence was not reported for the third hybrid dimer (‘27eS’, 2), our simulations predict that it should span two base pairs between covalently reacting G and A bases (e.g., 5′-GTAT(A)-3′).     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.