Indian Journal of Biochemistry & Biophysics Vol. 36, June 1999, pp. 177-1 87

Effect of sulphur crosslinking on the stability and transition of triple helical DNA

Seema Srivastava, Vishwambhar Dayal Gupta and Shyam Singh Divi sion of Biopolymers, Central Drug Research Institute, Lucknow 226 001 , India

Received 8 September 1998; revised 8 January 1999

In continuation to our work on order-order and order-disorder transition in triple stranded DNA when it is bounded to netropsin, we report in this communication the stabilizing / destabilizing effect of disulphide linkage on the phase dynamics of the triplex using the amended Zimm-Bragg theory. It is observed that in contrast to the sequential triplex ~ duplex ~ single strand melting of the uncrosslinked triplex, crosslinking causes the triplex state to melt directly to the single stranded state, with no apparent intermediary of a duplex state. Since there is no overall difference in the enthalpy of crosslinked and uncrosslinked triplexes, the transition is entropy driven.

It is well known that the nucleic acid molecules can associate to form a wide range of higher order structures like left handed and parallel-stranded duplexes, hairpins with ordered loops, triplexes, and quadruplexes, etc.I-IO . Many of these structures are stabilized by non Watson-Crick base-base and base-backbone. interactions. DNA triple helices are known to exist in two different forms. The two forms differ in the sequence composition of the third strand, the relative orientation of the backbones of the three strands and the base triple interactions. In both structural forms the third strand is bound to the major groove of a homopurine-homopyrimidine Watson­Crick duplex domain . In one class the third pyrimidine rich strand binds to the purine tracts in the major groove via specific Hoogsteen type hydrogen bonds and lie parallel to the purine Watson-Crick strand . In a second class the third purine rich strand binds to the purine tracts in the major groove of Watson-Crick duplex and lie antiparallel to the Watson-Crick purine strand forming reverse Hoogsteen hydrogen bonds.

These DNA triple helices are found in H-DNA which may exist in vivo and may play a role in mediating cellular events. The triple helix formation could serve as a general solution for the sequence specific recognition of double stranded DNA and RNA II-15

. Such recognition will be important in the development of reagents for the physical mapping of chromosomes and in the development of the anti gene therapeutic agents capable of site specific inhibition of transcription in vivo.

• To whom all correspondence may be addressed. Communication number 5828

Several research groups are trying to stabilize the secondary structures of nucleic acids by di sulphide bond crosslinkingI6

-25

. Efforts have also been made to use this modification to design DNA and RNA constructs for biophysical characterization and molecular recognition studies. The most common method to crosslink DNA duplexes is to link the DNA backbones by means of nucleotide hairpin loops or some chemical vanatlOns in the DNAs. The thermodynamic studies of such constrained model systems have provided unique insights into the forces that stabilize DNA structures l6

.

Considerable efforts have been made towards thermodynamic characterization of melting transitions . I' 'd I I 451626-33 Th " 'd In nuc elc aCI mo ecu es '" . IS provi es information about the stability and temperature dependent melting behaviour of these structures. It also enables us to define the relative contribution of various molecular forces in nucleic acid structures. From such studies we can also extract information about the dependence of stabilities of triple-helica l complexes on their double and single stranded components, as well as solution conditions.

Recently Volker et al. 16 have reported thermodynamic properties of a conformationally constrained DNA triple helix. They have characterized the thermal and thermodynamic consequences of incorporating a disulphide crosslink into the Hoogsteen strand of an oligonucleotide which is capable of being folded in order to form an intramolecular DNA-triple helix 16 (Fig. I) . Spectro­scopic and calorimetric methods have been used for this. They have performed thermodynamic studies on both the crosslinked and its uncrosslinked parent. The

178 INDIAN J. BIOCHEM. BIOPHYS., VOL. 36, JUNE 1999

Model for the Denaturat ion a f 'the uncross linked Oligonucleoti de

T T T T

T • T T .... ).-T c .... G-C T .. .. A-T ~ c+ .... G-C T .... A-T T .. .. A-T c+ .... G-C

f T-A T T T T

Intramolecular ~ Triple Helix >

e T

T

T e T

T T T . T

T ~ T ~-r G-C A-T G-c A-T A-T G-e T-A T T TT

Hairpin with dangling 3tail

Model for the Denaturation at the

T T T T

T 5' T T .... ·A-T C· .. .. ·G-C T .. · .. A-T C · .. .. G-C T · .... A-T T .... ·A-T c+· .... G-C ,y-s's-X - A

3 T T T T

cross·linked ~<:--Intramolecular >

triple Helix

T T T T T 5' T

T ~-T C G-C T A-T C G-C T A-T T A-T C (;-c 3,y-s,s-f-~

TT

[Cross linkedl L Hairp'ln j

T T

A A

G A

GA 5'

Coil

31

Te

T T e

TT G

T C

T T

A

crosslinked Oligonucleotide

T e T C T

TT T T T T T

TC 3¥-s·s-x T

G

t G A

!s' cross linked

coil (Lariat)

C T

C

8

II

Fig. I - Schemati c representation of the temperature· induced unfolding pathway of the uncrosslinked (panel A) and crosslinked triplex (panel B) . [In panel B, the hypothetical crosslinked hairpin intermediate state is shown in brackets to indicate that it is not detected experimentally. (Reprinted with permission From Biochemistry, 1997,37, 756·766. Copyright 1997 American Chemical Society)

SRIVASTAVA et af.: EFFECT OF SULPHUR CROSSLINKING ON STABILITY AND TRANSITION OF DNA 179

transitions observed were characterized by a cooperative absorbance change as a function of temperature. It has been observed that uncrosslinked DNA triplex melts in two steps consistent with the melting behaviour of other such triplexes. The crosslinking induces an increase in the thermal stability of the initial triplex such that the triplex and the underlying duplex, unlike the uncrosslinked triplex, melt in a single transition . Further it was observed that all these transitions are pH dependent. Biphasic melting of the uncrosslinked triplex becomes monophasic below pH 7.0 while the melting of the crosslinked triplex remains monophasic over the entire pH range.

It is generally surmised that the disulphide crosslinking stabilizes the DNA triplex. In the present communication an attempt has been made to understand the energetics of the phase transition with and without disulphide crosslinking. The absorbance and heat capacity measurements data of Volker et at. 16 have been used to check the model. It is based on the Zimm and Bragg34 theory of helix-coil transition which has been suitably amended to explain order-order transitions in DNA triplexes. The effect of crosslinking is reflected in the nucleation parameter and the corresponding A type anomalous nature of heat capacity which agrees with the experimental measurements wherever available.

Theory A long chain molecule capable of making

transition from one state (h I) to another state (h2) can

be represented as sequence of hlhlhla2h2h2h2h2alhlhl -------- where al and a2 are the nucleation parameters (border state) in going from one ordered state to the

other ordered state (hr~hl) and vice versa (hl~h2)' If Sl and S2 are the equilibrium growth parameters for the two ordered states then the transition matrix for the order-order transition can be written as:

... (I)

This transition matrix is an amendment to the Zimm and Bragg matrix for helix-coil transition34

.

The model consists of two phases which can make transition from one to the other through a border state with large expansion of initial nucleation energy. Both of these states are ordered states.

In order to obtain the grand partition function of

the chain we have to build - in the end effects. The initial state vector U which gives the contribution to the first segment is written as:

· .. (2)

where a' and a" are the parameters giving the interaction of end segments with the solvent when they are in states hi and h2' respectively . The partition function for an N-segment chain is given as:

Z=UMN-IV ... (3)

where the column vector V gives the state of the last segment,

III V=I I

III · .. (4)

Going through the matrix evaluation formalism outlined by Zimm and Bragg l7, we obtain an expression for the fraction of segments in state I:

fl =0 In zlNa In Sl . .. (5)

Solving equation 5 we obtain

fl = (\ + Brl {(S/AI)(OAl/os l) + B(sI /A2)(OA2/osl) + 11N[(sI /AI)(oA l/os l)+ B (s/A2)(oA2/0 sl)]} ,

· .. (6)

where AI = (Sl + S2X - b 1..2) I ( 1.. 1-': 1..2) , A2 = (Sl + S2 X - b AI) I (1..2 - AI), AI and 1..2 are the eigen values of M and B = (X2/X I )N( A21 A I) with ,

XI=SI AI, X2=SI 1..2, b=( \ +X-a2-aIX) 1 (I - al(2), and 'X = a"la' (SI/AI)(OAl/os l) = (X I - I )/(X I - X2),

(S/A2)(OA2/osl) = (X2 - I )/(X2 - X IXsl1 A I )(oAl /os l) = [s2(2a-1 + b- X-ba) + s( 1+ X-b + ba - 2aX)]1 [{(I-s)2+ 4aX }{s+X -bX2}]

(sI/A2)(oA2/0sl)=[s2(2a - 1 +b-X- ba) + s(1 +X - b+ba -2aX)]/[{(I - s)2+ 4aX } {s + X - bXd] a=ala2 and s=s l/s2

The total absorbance of the nucleotide is given by:

Absorbance = f IAI + (I - f l)A2 .. (7)

where AI and A2 are absorbances in first and second states.

The extension of this formalism for specific heat is straight forward. The specific heat is related to the molar enthalpy and entropy changes in the trans iti on from state I to II. From well known thermodynamic relations, free energy and internal energy are F=-KnT In Z and U = -T 2(0/07) (F/7), respective ly. Diffcren-

180 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, JUNE 1999

tiating intemal energy with respect to temperature we get the specific heat:

... (8)

where !1H is the molar change in enthalpy about the transition point, S is entropy which is equal to S=exp[(!1H/R){(I /1)-(I/TF)}], TF is the transition temperature, and

Of/os=[-{fl/(I + B)}(oB/os) + {(%S){(S/"-I) (O"-I/OSI) }/(l + B) + {(%s)[ {(Sl/ A,) (oAI/(osl)}/N} + {(oB/os)[(S/"-2)(O"-2/0SI) + (s/ A2N)(oAi(oslm + B[(%s){ (sl/"-2)(0"-2/0s,)} + {(0/oS){(S/A2)(oA2/(OSI)}/N}],

oB/os = NB[( I/X2)(oX2/os) - (1IXI)(oXI/os)] + B[ {I - b(oXI/os)} /(s + X - bX I)} - {I - b(OX2/0S)}/(s + X - bX2)}]

oX/os= 1/2+ {(s-I + 2O')/2{(s-I)2+ 4O'S}II2} OX2/0S= 1/2 - {(s-I + 20')/2 {(s-li + 40's} 112}

(% s) {(S/"-I )(O,,- / OSI) = O'(s + 1)/ {(s_I)2 + 40's} 312

(%s){ (SI/"-2)(0"-2/0S I) = - 0'( S + 1)/ {( s-I )2 + 40's} 3/2

(%s)[(s/ AI)(8A/os l) = [[(l-si + 4O's][s + X-bX2] [2S(2O'-I +b-X-bO')+ (I +X-b+bO'-2oX)] -[(s2(2O'-I +b -· X-bO')+s(1 +X-b + bO' - 2O'X)][(1 - si + 4O's][1 - b(OX2/0S)]

. 2 +(s+X-bX2)(2s-2+4O')]/[(I-s) +4O's] [s+ X-bX2]

(0/os)[(sl/A2)(oA2/8s l) = [(1 - si + 40'S] [s + X - bXd[2S(2O' - 1 + b - X - bO') + (I + X - b + bO' - 2O'X)]- [(s2(2O' - I + b - X-·bO') + s« I + X-b + bO'-2O'X)][( l_s)2 + 4O's][l-b(oX/os)] + (s + X-bX I)(2s-2 + 40')] /[( I-si + 40'S] [s+ x-bx,f

Results and Discussion

As discussed earlier Volker et 0/.16 have reported

the thermodynamic properties of an intramolecular triple helix containing two all thymine linker loops in which the Hoogsteen strand is covalently crosslinked to the underlying Watson-Crick hairpin duplex by means of a disulphide bridge. The properties are compared with those of the corresponding intramolecular triplex with no disulphide crosslinks. CD and calorimetric measurements have shown that at low temperatures both the crosslinked and uncrosslinked structures exist in solution as triplexes . However as the temperature is increased the uncross linked structure goes through a biphasic

transition i.e. first through duplex formation and then to a "less ordered" (extended single stranded) structure. However crosslinking causes the triplex state to melt directly to the final single stranded state (lariat like) without the formation of any intermediate state. Both the systems init ially showed negative CD absorption bands at 213-214 nm (characteristic of triple helices) and later on with increasing temperature, in the uncrosslinked sample there is an initial loss of the 213-214 nm negative band accompanied by an increase in intensity and slight blue shift of the 280 nm positive band. This is characteristic; of duplex formation. Subsequent formation of an extended single strand is characterized by a decrease in positive ellipticity at 277 nm. In case of cross linked structure increase in temperature causes the CD ~pectra to go directly to spectra consistent with a final single-stranded " lariat like" state. Similarity of the CD signals of the two "denatured" states reflect similarity of the base unstacking in both the molecules at the elevated transition temperatures. The extended single stranded state is a less ordered (not random coil) state and has been observed earlier in oligonucleotides which do not have a random distribution of base pairs but are rich in sequence lengths of a given base type. This has been confimled by CD measurements35 on T rich oligonucleotides which show the presence of a positive band at 275 nm and a negative band at 240 nm with a cross-over point at 255 nm.

In the present communication we have attempted to explain the transition and heat capacity profiles of sulphur crosslinked and uncrosslinked intramolecular DNA triple helix on the basis of the two state model of Zimm and Bragg which has been suitably amended for order~order transition. The two models adopted for order-order/less ordered transition in case of uncrosslinked and crosslinked triplexes are shown in Fig. I . All the input parameters i.e . enthalpy change around the transition point for a given chain length, transition temperature, and nucleation parameters are given in Table I and Table 2. The first two have been taken from the measurements of J Volker et a/. 16. The results obtained by using equations (7) and (8) for the uncrosslinked and crosslinked triplexes are shown in Figs. 2,3,4 and 5,6,7 respectively. The va lue of N=34 in Volker's DNA represents the number of purine and pyrimidine units which constitute the backbone (extended single strand) . But in triplex or duplex there are stacks of hydrogen bonds between purine and

SRIVASTAVA et af.: EFFECT OF SULPHUR CROSSLIN KING ON STABILITY AND TRANSITION OF DNA 181

Table I - Input parameters for the order-order transitions in uncrosslinked DNA triplex

Transition Triplex -tDuplex + " less Duplex -t extended Triplex-t extended Triplex-t extended single parameters ordered" dangling tail single stranded state single stranded state stranded state

pH 7.0 7.0 5.5 4.5 Tm (K) 293 .5 336.2 330.5 334.0 t'1H kcal/M bp 4.84 6.74 5.80 6.66 X 1.3 1.2 1.2 1.2

0'1 9.0x I 0-2 I.5 x lo-1 1.6x 1Q-1 6.5x I0- 1

0'2 2.0x I0-2 3.0xI0-2 8.0x I0-2 4.0x I 0-3

0' 1.8x I 0-3 4.5x I0-3 1.28x 10-2 2.6x 10-3

N 14 19 33 33 AI 0 0 0 0 A2 0.999 0.999 0.999 0.999 t.T(exp.) 7.18 7.5

t.T(theo.) 7. 18 7.5 706 4.77

t.T(exp.) and t.T(theo.) are experimental and theoretical half widths respecti ve l)(, X is end effect parameter.

Tab le 2 - Input parameters for the order-order transitions in disulphide crosslinked D A trip lex

Transit ion parameters Triplex -t extended Triplex -t extended Tripl ex -t extended single single stranded state single stranded state stranded state

pH 7.0 5.5 4.5 Tm (K) 328.5 345.5 35 1.8 t.H kcal/M bp 5.56 6.2 1 6.80 X 1.3 0.7 1.2

0'1 8.0x I0-2 2.5x I 0-2 2.5x 10-2

0'2 3.0x I 0-2 9.5x I 0-2 8.0x I0-2

0' 2.7x I0-3 2.25x 10-3 2.0x I 0-3

N 31 3 1 3 1 AI 0 0 0 A2 0.999 0.999 0.999 t.T(ex p.) 4.68

L\T( theo.) 4.68 4.26 4.06

L\ T( exp.) and t. T( theo.) are experimental and th eoretical hal f widths respect ive ly. X is end effect parameter.

pyrimidine base pai rs and the value of N depends on the lIumber of hydrogen bonds which are broken during the transiti on. It would be equal to 14 in case of triplex to duplex transition and since the C+-G-C base pair is capable of making five hydrogen bonds, three on one sid e and two on the C + side36 Therefore this number would become 19 in dupl ex to single stranded state transition, three add itional because of G-C and two because of the additional link between Adenine and Thymine at the foldin g end (see Fig. I). As for the monophasic transition in uncrossed and cross linked cases since there is a direct tran si ti on from trip lex to an ordered sin gle st randed extended state, the values of are 33 and 31 respective ly. Uncrosslinked triplex

As remarked earl ier at pH 7.0, the melting of

uncrossl inked triplex takes place in two steps, involving first the expu lsion of the third Hoogsteen strand and next the melting of the remaining Watson Cr ick duplex to form a "less ordered" extended single stranded state l 6

. It was further observed that this two step melting of ON A triplex becomes monophasic below pH below 6.5 (experimental data reported only for pH 5.5 and 4.5) which means that the triplex melts direct ly into a sin gle stranded state without form ing the intermediate state. Unlike other triplexes' 1.32, the triplexes formed by intramolec ular hydrogen bondi ng on transition first goes into a duplex having hairpin structure with dangling 3' ta il. In the next step, the dup lex goes into a less ordered s ingle strand (Fig. I). Therefore the transition from triplex to duplex and from duplex to s ing le strand are all order-order

182

Q) u c ro .0 ..... 0 (/)

.0 ro c Q) OJ c ro

.J:: U

2

1.5

0.5

o 270

INDIAN J. BIOCHEM. BIOPHYS. , VOL. 36, JUNE 1999

~ 4

~ 3.5

~ 3 62.5

E 2 .. g. 1.5 U 1 tV ~ 0.5

o ) ' j _\'~.,

270 290 310 330 350 370

Temperature (K)

290 310 330 350 370 Temperature (K)

Fig. 2 - Transition profiles and corresponding heat capacity variation (shown in inset) for order-order transitions in uncrosslinked DNA triplex at pH 7.0 [Calculated values, (--), experimental values, (n.)].

1.2 ..,---------------- ----

Q)

g 0.8 ro .0 ..... o (/)

.0 ro 0.6 c Q) OJ C

~ 0.4 U

0.2

~ 10 j :.;: 9 '0 8 :§ 7 til

~ 6 ~ 5 'u til 4 c. <3 3 iii 2 Q)

I

o 270 290 310 330 350 370

Temperature (K)

-:.-----

o+---~~~~~~------~------~-----~~ 270 290 310 330 350 370

Temperature (K)

Fig. 3 - Transition profiles and corresponding heat capacity variation (shown in inset) for order-order transitions in uncrosslinked DNA triplex atpH 5.5 [Calculated values, (--) , experimental values, (u.)].

SRIVASTAVA et af.: EFFECT OF SULPHUR CROSSLINKING ON STABILITY AND TRANSITION OF DNA 183

16 1 :x;: 14

(5 12 .§ ~ 10

Q) 0.8 ~ 8 u ?:-

c:: Tj 6 ro cu

.0 Q.

..... cu 4 0 u I/) 0.6 iii .0 2

c:{ Q)

I c:: 0 Q) 270 290 310 330 350 OJ c:: 0.4 Temperature (K) ro

..r:::. U

0.2

O+---~~~====~~~~~--~------~------~ 270 290 310 330 350 370

Temperature (K)

Fig. 4 - Transition profile~ and corresponding heat capacity variation (shown in inset) for order-order transitions in uncrosslinked DNA triplex at pH 4.5 [Calculated values, (--) , experimental values, ( ... )].

10

:x;: 9

0 8 .§ 7

0.8 ~ 6 Q) ~ u ?:- 5 c:: U ro co 4 -e a.

III 3 0 u I/) 0.6 .0 iii 2 ro QJ

c:: I '. ' . 0

....... '.

w Ol

300 310 320 330 340 350 c:: 0.4 ro ..r:::. Temperature (K) U

0.2

o+-----~~~----~~~~~------~------~ 260 280 300 320 340 360

Temperature (K)

Fig. 5 - Transition profiles and corresponding heat capacity variation (shown in inset) for order-order transitions in crosslinked DNA triplex at pH 7.0 [Calculated values, (--), experimental values, ( ... )).

184

1

Q) 0.8 u c C'Il -e o

~ ~~ j o 16 J; 14

,[ 12 I .~ 10 1 ~ 8 l "' 6 ~ U I i;j 4 1 ~ 2 '

INDIAN J. BIOCHEM. BIOPHYS., VOL. 36, JUNE 1999

.2 0.6 O+-- - ==:;==------ ---"=="'T------j

C'Il

c Q) CJ)

iij 0.4 .c u

0.2

310 330 350

Temperature (K)

370

~ o +-----.-~~~~~ .. ~.~ .. ~ .. ~ .. ~.-_,------r_-----r_--

310 320 330 340 350 360

Temperature (K)

370 380

Fig. 6 - Transition profi les·and corresponding heat capacity variation (shown in inset) fo r order-order transitions in crosslinked DNA triplex atpH 4.5 [Calculated values, (--), experimental values, ( ••• )].

Q'18 r .:... 16 0

~ 14 \ rl 12 I .x :; 10

Q) 0.8 (3 8 ro U Q. 6 c ro C'Il 0

4 -e ro Ql 2 0 I

Cfl 0.6 .D 0 C'Il

310 330 c 350 370

Q) Temperaturp. (K) CJ) c 0.4 C'Il .c U

0.2

o +-----~--==~~~~-----~------~----~----~ 310 320 330 340 350 360 370 380

Temperature (K)

Fig. 7 - Transition profiles and correspond ing heat capacity variation (shown in inset) fo r order-order tran siti ons in crosslinked DN1\ trip lex atpH 4.5 [Calculated values, (-- ), experimental values, ( • •• )].

SRIV ASTA V A et al.: EFFECT OF SULPHUR CROSSLINKING ON STABILITY AND TRANSITION OF DNA 185

transitions and hence the theory of order-order transition has been applied at both the stages. Applications of order-disorder transition leads to results which are far removed from the experimental measurements. These transitions can occur as a function of temperature and other thermodynamic parameters like pH etc. The deviation of the end effect parameter from unity (for a finite chain) is also shown. These end effect parameters play partly the role of scaling factors and may have their origin not only in" the finiteness of the chains but also in the nature of interactions and experimental measure­ments. The value of N depends upon the number of hydrogen bonds broken base pairs. Our calculations correspond to N = 14, 19, and 33 for the Triplex ~ Duplex + " less ordered" dangling tail, Duplex ~ extended single stranded state (at pH 7.0) and Triplex ~ extended single stranded state (at pH 5.5 and 4.5) respectively. On changing the pH values from 7.0 to 5.5 and 4.5, the alteration in the melting pathway (biphasic to monophasic) of DNA triplex is an indication of the increase in stability such that the triplex melting occurs at a temperature where the intermediate duplex state is no longer thermally stable, thereby resulting in the transfonnation of the triplex directly into the single stranded state l6 below pH 7.0. This is a result of the enhanced cooperativity and consequent stability as happens in case of disulphide linkage. In other words lowering pH and introducing disulphide linkage result in a new condensate state with enhanced cooperativity arising possibly due to enhanced non-bonded and ionic interaction.

From the smaller value of nucleation parameter and larger change in enthalpy at pH 4.5 in comparison to pH 5.5 it is clear that the triplex to extended single stranded state transition at pH 4.5 is sharper or more cooperative than the corresponding transition at pH 5.5. This observation is again consistent with the pH induced increase in trIplex thermal stability as a result of which the DNA triplex melts at a higher temperature at pH 4.5 in comparison to pH 5.5. The increase in the stability of DNA triplex at lower pH is due to greater hydrogen ion concentration at lower pH. This results in intramolecular ionic interactions and a new condensate state which has higher cooperativity and enhanced stability. All the theoretical transition curves are in very good agreement with the observed experimental data of Volker et al.16 . The nucleation parameters which provipe the best fit for the transition profiles also

successfully generate the A-point heat capacity anomaly shown in Fig. 4. Thus the model adopted and the theoretical approach developed successfully interpret the transition and heat capacity profiles.

Disulphide crosslinked triplex It has been observed that the disulphide crosslinks

effectively stabilize the DNA triplex, thus the melting pathway of the DNA triplex is altered and it melts directly to a constrained "lariat-like" final single stranded state l6

. The stabilization is a result of increase in the configurational interaction because the overall enthalpy change in transition from the triplex to a singlet state remains unchanged. In other words, the transition is entropy driven rather than enthalpy driven. e.g. at pH 7.0 for the crosslinked triplex to singlet transition !:!.H = 87.5 kcall M and for the

uncrosslinked one at the same pH the total enthalpy change is equal to 33.9 + 53 .9 = 87.8 kcal/M. Here again the second state is an ordered state, hence the theory of order-order transition has been applied to explain the triplex to single stranded state transitions in crosslinked triplex. The results on the order­disorder transition are again too far off. All the transition curves are shown in Figs. 5-7. Depending on the interacting base pairs, the value of N has been taken to be equal to 3 I in all transitions. As mentioned earlier, various transition parameters, i.e. enthalpy change around the transition point for a given chain length , transition temperature, and nucleation parameter are given in Table 2.

Volker et al. IG have observed that on lowering pH the stabilty or the DNA tTiplex goes on increasing. Thi s is reflected in the lower value of the nucleation parameter. In turn it is a reflection of greater cooperativity I stability and sharpness of transition at lower pH. The nucleation parameters are 2.7 x 10.3,

2.25 x 10·:1 and 2.0 x 10.3 at pH 7.0, 5.5 and 4.5, respectively. Their dependence on crosslinked and uncrosslinked systems is given in Table I. Lower the nucleation parameters, greater the cooperati vity. Further these results are consistent with the larger enthalpy change at lower pH values. At pH 7.0, 5.5 and 4.5 the corresponding enthalpy changes are 5.56, 6.21 and 6.80 kcal/mole base pairs .

To summarise, comparing our results on cross­linked structure with those on uncrosslinked structures, it is clear that disulphide crosslinking stabilizes the structure better at the same pH. This added change in the stabi li ty is reflected in the values of the nucleation parameter and enthalpy change. The

186 INDIAN J. BJOCHEM. BlOPHYS., VOL. 36, JUNE 1999

former shows a decrease and the latter an increase. Our results are also supported by the experimental observations ofYolker et a1. 16

•

Heal capacity Heat capacity is an important thermodynamic

quantity which characterises the conformational and dynamical states of a macromolecular system. Such . measurements for disulphide crosslinked DNA triplex­and its uncrosslinked parent are also reported by Volker el al. 17

• Theoretically, it is proportional to the second derivative of the Gibb's free energy and has been calculated by using equation (8). The heat capacity curves with A point anomaly for various transitions are shown as inset in Figs . 2-7 along with the transition profiles. Wherever available the experimental data points are also shown. The theoretically obtained heat capacity profiles agree with the experimentally reported ones and could be brought almost into coincidence with the use of scaling factors. These factors take into account any constant error in experimental measurements which causes a constant shift of the experimental curves . This can also be said of the theoretical modelling which is essentially based on a two state model whereas in practice the model must consider multiphasic presence. The sharpness of the transition can be characterized by the half-widths at half maximum of the heat capacity curves given in Table I and Table 2. The experimental half widths are in good agreement with the theoretical ones. It is observed that the half widths of the order-order tran sition in crosslinked and uncrosslinked DNA triplex at pH 4.5 are smaller than the corresponding transitions at pH 5.5 and 7.0 . This shows greater sharpness of transition and hence stability at lower pH value as described earlier. Further it is observed that the transition half widths of the crosslinked structure is smaller than the corresponding transition in uncrosslinked structure. This is a result of the disulphide bond which contributes to the stability of the triplex . It has been noticed that during Triplex ~ Duplex + " less ordered" dangling tail transition, the heat capacity max imum has a smaller value as compared to the duplex ~ singlet (extended) transition . This is due to the larger enthalpy change in the second transitions involving dupl ex ~ singlet states as compared to the first step Cfable I) . Since the "extended" state is not a unique ly defined state, the heat capacity differences could also arise due to the presence of short helical segments present in this state. Similarly because of enhanced

cooperatiyity the heat capacity of the cr6sslinked triplex is always higher than the heat capacity of uncross linked triplex. As the heat capacity is sensitive to low frequency modes, this could possibly be due to the larger cooperatiyity , implying longer cooperative length and larger number of lower frequency modes.

As for the disagreement between the experimental and theoretical curves, in some cases near the turning points, one always aims at the best optimization to. interpret the experimental data. In other words, the best possible agreement should be obtained in all parts of the transition profile and not only in some parts of it. It is from this point of view that the totality of the theoretical interpretation has been attempted. Part of the disagreement near the turning points (h igh temperature) may lie in the extrapolation/ interpolation of the experimental data and errors of measurements.

The agreement With the heat capacity data, which is related to Ithe derivative of the transition profile, is excellent in almost all cases The A-point anomaly in heat capacity is also cooperativity dependent. Thus in the interpretati~:)11 of the experimental data one has to look for the best interpretation of the entire data. In the present case the cooperatiyity index is adjusted to give the best fit of both the transition and associated heat capacity data.

Conclusions The phase tranSitIOn In the intramolecularly

stabilized and . disulphide crosslinked and uncrosslinked triple helical DNA can be successfully interpreted within the theoretical framework of Zimm and Bragg34 using only two variable parameters; namely, the nucleation parameter and the propagation parameters (scaling factor being very close to one). The consequences of pH variation, concentration dependence of stability, ligand binding, salt condensation crosslinking etc. are all contained in these two parameters. This is, in fact, the strength as well as the limitations of the Zimm-Bragg approach . The total ity of theoretical interpretation has been attempted in all parts of the transition profile. Half widths are inverse indicators of the extent of cooperativity. A very good agreement is obtained between the half widths of experimental and theoretical transition profiles.

Acknowledgement Financial assistance to VDG and SS from Council

of Scientific and Industrial Research, New Delhi

SRIVASTAVA et al.: EFFECT OF SULPHUR CROSSLINKING ON STABILITY AND TRANSITION OF DNA 187

under the Emeritus Scientist Scheme and Research Associateship respectively IS gratefully acknow Iedged.

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