α-DNA IV: α-anomeric and β-anomeric tetrathymidylates covalently linked to intercalating...

volume 15 Number 16 1987 Nucleic Acids Research

a-DNA IV: a-anomeric and /S-anomeric tetrathymidylates covalently linked to intercalatingoxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding

Claudie Gautier, Francois Morvan1, Bernard Rayner1, Tam Huynh-Dinh2, Jean Igolen2, Jean-LouisImbach1, Claude Paoletti and Jacques Paoletti*

Laboratoire de Biochimie, INSERM U 140, CNRS LA 147, Institut Gustave Roussy, 94800 Villejuif,•Laboratoire de Chimie Bio-organique, UA 488 du CNRS, Universite des Sciences et Techniques duLanguedoc, 34060 Montpellier Ce"dex and 2Unite" de Chimie Organique, ERA/CNRS 927, InstitutPasteur, 28 rue du Docteur Roux, 75015 Paris, France

Received May 18, 1987; Revised and Accepted July 25, 1987

f ABSTRACT.7$ new set of molecules made of an intercalating agent (oxazolopyrido-carbazole, OPC) covalently linked through a polymethylene chain of variouslength to the S' end of o-anomeric or p-anomeric tetradeoxynucleotides (o-or p-T*) have been synthesized. The p-thymidylate modified compound (p-T4C5OPC) is able to interact with the complementary sequence, p-poly (rA) ;this interaction is strongly stabil ized compared to the parent compound, p-o l igo(dT)^ and is specific for poly (rA). The molecule synthesized from the

' unnatural a-anomer, a -TÔPC, is also able to in te rac t with poly (rA)leading to the formation of an a-p hybrid stabilized by the energy providedby the OPC moiety. The stoechiometry of the Dinding reaction shows that anA-T pairing occurs in the a-p heterohybrids. Tm studies reveal that the a-pheterohybrids are more stable than their p-p counterparts.

INTRODUCTION.

The regulation of gene expression in both procaryotes and eucaryotes

requires the specific recognition of single-stranded or double-stranded

nucleic acid base sequences. Usually, this regulation is controlled by

sequence specific proteins. In some case, however, regulation is achieved

through a nucleic acid fragment. Recently such fragment was found in

procaryotes as a repressor RNA complementary and therefore selectively bound

to target messenger RNA (1).

Recent progress in the chemical synthesis of oligonucleotides has permit-

ted the development of oase sequence specific molecules designed to regulate

specific viral or cel lular gene expression (2). Such oligonucleotides have

already been used to alter translation or the coupling between transcription

and translation in bacteria (3). Modified oligonucleotides have also been

synthesized with methylphosphonates instead of phosphates in the internucleo-

t id i c linkage and have been shown to modified viral gene expression (4). More

recently, new series of base sequence specific oligonucleotides has Deen

designed in such a way that their binding a f f in i t y was reinforced by the

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Nucleic Acids Research

covalent l inking of an intercalating agent (5-8). Such molecules have been

found to show interesting biological properties related to the control of

gene expression (9-10).

A major problem related to in v i t ro or in vivo u t i l i sa t ion of such mole-

cules is their poor stabi l i ty toward nucleases degradation (11-12). Besides

modifying the phosphodiester linkages in order to improve their s tab i l i ty

(13), another original way to obtain stable compounds, is to synthesize

unatural oligodeoxyribonucleotides consisting exclusively of o-anomeric

deoxyribonucleotide units. Preliminary studies have established that these a-

oligonucleotides might be very poor substrates for a l l kinds of nucleases

since they are completely resistant to calf spleen phosphodiesterase and at

least ten fold more resistant toward degradation by snake venom phospho-

diesterase tnan their p counterparts ( lb-17). In addition, tnese new deriva-

tives retain an ab i l i t y to reanneal with their p-complement (14) as predicted

from a study on Ureiding stereomodels ( l b ) . The a-p hybrids are made of

parallel strands as f i r s t suggested by Sequin from theoretical considerations

on i)reiding stereomodels (15) and experimentally confirmed oy NMrt studies

(J.W. Lown, personal communication) and foot-pr int ing experiments (C. Helene,

personal communication).

These findings prompted us to design an a-oligonucleotide linked to a

DNA intercalating agent. We expected to obtain new compounds endowed with

both properties of increased binding a f f in i t y to a complementary strand and

increased resistance towards nucleases. In addition, this work might open

the way to research oriented toward new nucleic acids structure which might

be of interest from the t r ip le point of view of their structures, their

pnysico-chemical properties and their pharmacological potent ia l i t ies, even

i f they have so far not Deen reported to exist in nature.

We report here the synthesis and some physicochemical properties of these

new model compounds as well as their p counterparts. OPC derivatives nave

been selected here because these f l a t molecules which have been thoroughly

studied (18-20), do intercalate into UNA, this process being accompanied by

an increase of fluorescence (21).

Using absorption and fluorescence spectra, we confirm that such modified

oligodeoxyriDonucleotides are able to form hybrids with complementary p-homo-

polyribonucleotides, and tnat the o-thymidylate-p-ribonucleotide hybrids are

more stable than the p-thymidylate-p-ribonucleotide derivatives.

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MATERIALS AND METHODS.

General methods.

Mass spectra were recorded on a VG 70-250 mass spectrometer equipped

with a FAB gun.

Ultraviolet determinations were obtained using a thermostated Uvikon

810 spectrophotometer (KONTRON) equipped with a temperature attacnement.

Fluorescence spectra were recorded on a thermostated SFrt 25 spectrofluoro-

meter (KONTRON).

Amino acids were purchased from Sigma and were protected by a dimethoxy-

t r i t y l group as described by Hernandez and c o l l . (22). 2-methyl-9-hydroxy-

e l l ip t ic in ium was obtained from SANOFI and horse radish peroxydase was from

SIGMA. The interaction determinations were usely performed in Cacodylate

buffer, 0.1 M, pH 7, NaCl 1 M. Any change from these conditions w i l l be ind i -

cated in the text.

High performance l iquid chromatography was made on a Perkin Elmer appa-

ratus equipped with LC75 Perkin Elmer UV detector.

Poly(rA) was purchased from Boerhinger (Manheim) Lot 10421221-18.

Synthesis.

A detailed discussion of the methods of synthesis w i l l be presented else-

where (manuscript in preparation).

This synthesis is achieved through three major steps.

(1) Synthesis of properly protected a- or p-tetrathymidylate derivatives

retaining a free 3'-OH end.

(2) Addition of a linker of chosen length to the 3'-OH end of a- or p-

T4 oy formation of an ester linkage at the -COUH terminal end of a linear

aminoalkyl acid.

(3) Condensation of the el i ip t ic in ium derivative moiety, on the free -

NH2 end of the intermediate previously synthesized. This condensation is

obtained through peroxidase -H2O2 oxidation, in experimental conditions

already published (18) and leads to an oxazolopyridocarbazole ring (OPC).

The most noticeable features of the experimental set up and chemical

determinations are as follows (figure 1) :

5'-0 Dimethoxytrityl-p- and g -T t r i e thy l ammonium salt : ^ and ^ .

The f u l l y protected p-T (0.200 g - 0.10 mmoles) was treated by a solu-

t ion of 0.3 M TMG PAO (tetramethy 1 guanidinium pyridine-2-aldoximate) in

water-dioxane (1 /1 , v/v, 20 ml) overnight at room temperature. The mixture

was evaporated to dryness and the residue was dissolved in 30 ml of aqueous

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R v OHior-Mo]0

A .

DMTr T_T_T_T - f - T.TXT.1_: Anomer2:Anomer i

_l_l_Oc[cH2]nNH2 2 : Anomer

4 : Anomer ot , n= 5

a: n = 5b n = 7e n = 10

a: n = 55^- Anomerp b:n = 7

c n = 10

6 : Anomer Ot , n = 5

Figure 1. Major steps in the synthesis of a and p tetrathymidylate cova-lently linked to an OPC derivative through a polymethyiene linkage.A and B : p and a thymidine. C : 2-methyl-9-hydroxye11ipticinium acetate.D : N-dimethoxytritylaminoacid.Reactions conditions ( i ) : N-dimethoxytritylaminoacid and dicyclohexyl-carbodiimide/pyridine ; ( i i ) : Acetic acid SO % ; ( i i i ) : £ and horse-radishperoxydase/H202/0.02 M phosphate buffer pH 7.

ammonia. The resulting solution was heated at 5U°C for 5 hours, cooled and

evaporated to dryness. The residue was washed with diethylether and then

chromatographed on a Lichroprep RP 1H column [l.d x lu cm) using a stepwise

gradient of methanol (0 to 30 %) in 50 mM t r i ethyl ammonium hydrogeno-

carbonate. The appropriate fractions were comDined and evaporated to dryness.

Lyophilisation of the residue afforded the desired compound i_ as a colorless

powder (0.150 g) in 94 % y ie ld .

Unblocking of the internucleotidic phosphate groups of the fu l ly protec-

ted a-T- (0.393 g - 0.15 mmole) was achieved by a solution of 1 M TMG PAO in

water-dioxane (1/7, v/v, b ml). The mixture was heated at 70°C for 9.b h,

cooled and evaporated to dryness. The residue was purif ied as described for

compound 1_. The desired compound Z_ was obtained as a colorless powder

(0.223 g) in 84 % y ie ld .

Synthesis of 3' modified tetrathymidylates : 3_ and 4̂ .

To a so lut ion of 5 ' -0-d i methoxy t r i t y l - T 4 J. or ^ (0.150 g - 0.10 mmol)

and N-dimethoxytrityl aminoacid (D) (n = 5, 7 or 10) (0.50 mrnol) in anhydrous

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pyridine (5 ml) was added dicyclohexylcarbodiimide (0.206 g - 1.0 mmol) and

N,N-dimethylamino-4-pyridine (0.061 g - 0.50 mmol). The resulting solution

was st i r red for 6 h at room temperature. Water (1 ml) was then added and the

mixture was f i l t e red . The f i l t r a t e was evaporated to dryness and the residue

was precipitated from diethylether (this last operation was repeated three

times). The precipitate was dissolved in THF-methanol (7/3, v/v) and was

chromatographed on Sephadex LH 20 column. Fractions containing the product

were pooled and evaporated to dryness. The compound was then dissolved in

80 % acetic acid (5 ml) and the solution was kept at room temperature for 20

min. To the residue was added toluene and the resulting mixture was evapo-

rated to dryness (this operation was repeated several times). The residue was

washed by diethylether and then purif ied by chromatography on Lichroprep

RP IS column (1.2 x 10 cm) using a stepwise gradient of methanol (0 to 40 %)

in water. The fractions containing the product were evaporated to dryness.

The product was treated with Dowex 50 irf (NH4+ form) and lyophil isated.

Compounds .3 t 0 . i w e r e obtained as a colorless powder in 49 to 65 % y ie ld . The

purity of the products was checked by HPLC on Nucleosil C18 using a gradient

of acetonitr i le in o. l M t r i ethyl ammonium acetate pH 7. I t was superior to

99.5 %.

3_a Mass : 1268.69 : [M+H]+ UV(in water) \ : 207nm, 267 nm

3b Mass : 1297.27 : [M+2H]+ ; 1318.37 : [M+Na]+ ; 13J4 : [M+K]+

UV(in water) \ : 207 nm, 267 nm

2c Mass : 1338.44 : [M+H]+ UV(in water) \ : 207 nm, 267 nm.

Synthesis of the tetrathymidylate covalently linked to the e l l i p t i c i ne der i -

vative (c) : 5_ and (>.

To a solution of compounds 2 o r i . (0.U46 ram°l) and 2-methyl-9-hydroxy-

el l ip t ic in ium acetate (0.008 g, 0.023 mmol) in 0.02 M phosphate buffer (pH 7,

40 ml) was added horse-radish peroxydase (0.8 mg). After dissolution of the

enzyme, 20 mM hydrogen peroxyde (1.2 ml) was added dropwise. The solution was

f i l te red and evaporated to dryness. The residue was then prepurified by

chromatography on reverse phase Lichroprep RP 18 column using increasing

proportions of methanol (0 to 60 %) in water. Fractions containing the f luo-

rescent compound were pooled and evaporated to dryness. Purif ication was

achieved by high performance l iqu id chromatography on Nucleosil C18 column

using a linear gradient of acetonitr i le (0 to 60 % in 20 min) in 0.1 M t r i -

ethylammonium acetate, pH 7. Appropriate fractions were evaporated to dryness

and lyophil i sated. The product was treated with Oowex 50 W (NH4+ form) and

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Table 1 . Extinction coefficient and thermodynamical parameters corresponding

to self-association of the tetrathymidyiate modified compounds.

Compounds ^ ^ ( M - ^

a t 8°C

B ' l ) e 310nm ( r t ' l c n r l

at 8°C 8°C

A~*) Thermodynami c

parameters

p-T4C50PC e = 53500M

eD = 26500

e = 41800M

eD = 32000

2.2 105 AH° = -8.7 k c a l /

mole

AS" = -6.2 ue

AG°(281°K) = -7.0

kcal/mole

= 50000

eD = 26000

eu = 39200 1.25 105 AH° = -7 .b kca l /M

mole

= 29MW AS° = -4 .1 ue

AG°(281<>K) =-b.45

kcal/mole

r = b9000M

eQ = 23500

eu = 44000 4.5 105 AH° = -7 .6 kca l /M

mole

AS" = -1 .4 ue

Ab"(281<>K) = -7.2

kcal/mole

eQ = 27400

Derived parameters corresponding to a dimerization model for a- and p-thymidylate-OPC derivatives.

extinction coeff ic ient of the monomeric form£„extinction coeff icient of the dimeric form

K = dimerization constant.These values were calculated according to the theory of Schwartz et a l . (30).

lyophil isated. Compounds ^ (a) to (c) and j> were obtained as a colorless

powder in a y ie ld varying from 5 to 12 %.

J>a Mass : 1576.22 : [M+K]+. OV (in water) : 271 nm (e = 47800) ; 318 nm (e

= 47000). Fluorescence : \exc : 318 nm, x.em : 525 nm.

jto Mass : 1567.42 : [M+H]+ ; 1590 : [M+H+Na]+ ; 1611 : [M-H+2Na]+ ; 1634 :

[M-H+3Na]+. UV (in water) : 271 nm (e = 45000) ; J18 nm (e = 444U0).

Fluorescence : xexc : 318 nm, \em : 525 nm.

5c Mass : 1630.82 : [M+Na]+ ; 1653.51 : [M+2Na-H]+ ; 1675 : [M+3Na-2ri]+;

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50

2 45

IEN

T

t 40DU

1IO

N

u? 35—X

LU

A

I\\

i i i10 20 30

CONCENTRATION (xtO6M )

40

TEMPERATURE

60

Figure 2. A : Concentration dependence of apparent extinction coeff ic ient .The apparent ex t inc t ion c o e f f i c i e n t e.^p = Ao17/Co is shown as a functionof total (Z-T4C5OPC ( - • - ) or total P-T4C5OPC ( -0- ) concentrations, for samplesin 10 m M cacodylate buffer pH 7 and 0.1 M NaCl at 9°C.B : Temperature dependence of apparent extinction coeff icient.- • - C1-T4C5OPC-o- R-T4C5OPC

1697.12 : [M+4Na-3H]+. UV (in water) : 265-270 nm (e = 29500) ; 317 nm

(e = 38700). Fluorescence : xexc : 315 nm ; xem : 530 nm.

6 Mass : 1561.44 : [M+Na]+ ; 1583.67 : [M-H+2Na)+ ; 1605 : [M-2H+3Na]+.

UV (in water) : 271 nm (E = 46500) ; 317 nm (e = 44000). Fluorescence :

Xexc : 318 nm, Xem : 525 nm.

RESULTS.

Self-association of a and p tetrathymidyiate-OPC.

The absorption characteristics for each compound are shown on Table 1

and compared to the oxazoiopyridocarbazole moiety (OPC) alone. I t appears

that the maximum of absorption of the OPC is shifted toward the higher wave-

length once linked to the oligonucleotide residue. The UV spectra of a-and p-

T^CgOPC are s i m i l a r . Furthermore the data indicate a strong solvent ef fect

suggesting that self-association might take place. I t has been demonstrated

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0 4

3 03z

AB

SO

RB

s

0.1

g

• A .

i f w ly y\

250 300 350

WAVELENGTH (nm)

400 250 300 350

WAVELENGTH (nm)

4 0 0

0 2

AB

SO

RB

AN

CE

O

j , 00^ ^ — - 0 3

"A Ai V\

\1

c

250 300 350

WAVELENGTH (nm)

4 0 0

0.4

0.3UJJZ•a

3 0.2D

0.1

D

W \I//lKw m\

i i i ^ " ^

250 300 350

WAVELENGTH (nm)

400

Figure 3. Changes in absorp t ion spectra of p-T4Cn0PC ( 1 0 " 5 M ) and a-T4C5OPC in the presence of increasing concentrations of poly (rA) at 9°C ina pH 7 buffer containing 10 m M sodium cacodylate and 0.1 M NaCl. The valuesrepresent ratios of nucleotide over OPC concentrations.A : p-T4C50PC, B : p-T4C70PC, C : p-T4C100PC, 0 : a-T4C50PC.

that e l i i p t i c ine derivatives were able to self-associate in solution (23) as

other intercalating agents (24-29). These papers strongly suggest that the

same parameters which control this self association, also control the inter-

action with ONA. I t was then of interest, in the case of the a- and p-

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thymidylate-OPC compounds, to study i f any self-association was s t i l l main-

tained once the OPC ring was linked to the oligonucleotide. There is a strong

concentration dependence of the apparent extinction coeff icient of the a- and

p-thymidylate-OPC compounds (figure 2). Upon d i lu t ion, the apparent extinc-

t ion coefficient of the drug solution increases at the aDsoroance maximum at

odds with the Beer's law. Furthermore, we have also plotted the temperature

dependence of the apparent extinction coeff ic ient. By assuming a simple

dimerization model, we can apply the Schwartz and al theoretical treatment

(30) and derive the dimer extinction coefficient and the dimerization cons-

tant of the compounds. The results of such treatment are shown on Table 1,

together with the derived thermodynamic parameters. The p-T^ÔPC molecule

does not f i t the simple dimerization model and therefore no data are reported

concerning this compound.

Interaction of p-T.C OPC with homopolynucleotides.

In terac t ion of p-T.C OPC with different polyribonucl eoti des was investi-

gated using absorption spectroscopy. Only poly (rA) gives rise to modifica-

tions in the absorption spectrum of the molecule. No interaction was detected

with non complementary poly (rU) or poly (rC). Furthermore, this interaction

is l imited to single-stranded homopolynucleotides since no interaction is

detected with double-stranded poly(rA-rll). This indicates that the oligo-

nucleotide linked OPC moiety is unable to intercalate between base pairs of a

double-stranded structure contrary to what is observed for OPC alone. This

has been confirmed by a lack of interaction (UV absorption, fluorescence

exc i ta t ion) of any T.C OPC with calf thymus ONA. Poly (rA) was then used for

the interaction studies. Al l experiments were performed at pH 7 in cacodylate

buffer 0.01M, in order to avoid any double helix formation by poly (rA).

Fig. 3 shows the changes in the absorption spectrum induced upon binding

of p-TÔPC (n = 5,7 or 10) to poly (rA) at 9°C. At 270 nm, the reannealing

of the oligonucleotide moiety can be followed whereas the binding of the OPC

ring is followed at 310 nm, a region of the spectrum where the oligo-

nucleotide residue does not contribute to the absorption. This binding at 310

nm is characterized by an hypochromic sh i f t ; i t is accompanied by a batho-

chrome e f f e c t . p-T4Cg0PC binding yields two isobestic points at 324 nm and

347 nm ; on the other hand, the 324 nm isobestic point is not apparent after

the binding of p-T4C7OPC. These observations w i l l be br ie f ly discussed below.

Almost no changes are detected when p-T.C1QOPC is mixed with poly (rA).

A plateau is observed for concentration ratios of poly (rA) and T C OPC

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0.7 -

7A.0 S

1.0

0.9

0.8

0.7 -

1

10

B

n °

I

0 5

1.0

0.9

0.8

0.7

\

A

10

0

10 ,TÔPC ÔPC

Figure 4. Relative absorption of P-T4C5OPC (A), P-T4C7OPC (B), P-T4C100PC(C) and 0-T4C5OPC (D), in the presence of increasing concentrations ofpoly (rA) at 270 nm (•) and at 310 nm (D) for A.B.C and 0. Same determinationfor H-OPC (A) at 300 nm (A).Same experimental conditions as in figure 3.

higher than 4 and the stoichiometry of the binding corresponds to the forma-

tion of a number of A-T base pairs equal to the number of thymines in the

modified oligonucleotide (Fig. 4) . This stoichiometry of 4:1 has been derived

by determining the intercept between the slope of A/A° = f(poly(rA)/OPC) and

the plateau. Such a determination has been jus t i f i ed by a complete study of

the interact ion, taking into account the competition between the autoassocia-

t ion of the compounds and their binding to poly(rA) (manuscript in prepa-

rat ion) , according to Schwartz et al (30). Such detailed study leads to

values of the stoichiometry of 3.8 for both p-T4Cs0PC and p-TÔPC. T^ÔPC

does not show any effect at 270 nm indicating that no annealing takes place

between this compound and poly (rA), in these conditions.

The associat ion of p-T.CgOPC to poly {rA) induces an augmentation of

the fluorescence of the OPC r ing ( \ and \ . equal to 318 nm and 525 nm

respectively). Fig. 5 shows the variations of the fluorescence intensit ies

when poly ( rA) , 1 0 - 5 M , is t i t ra ted with increasing amounts of p-T4Cn0PC. We

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100 -

0 1 2 3 4

[T4C5OPC] (HM)

Figure 5. Fluorescence in tens i ty of p-T4C50PC bound to poly rA (10 " 5 M) forvarious T4C5OPC concentrat ions. Measurements were carr ied out at 9°C in10 m M sodium cacodylate/0.1 M NaCl, pH 7. Excitation and emission wave-lenghts are 318 and 525 nm respectively. The open triangle corresponds to thefluorescence of p-TÔPC and p-T4C,00PC ( I t - I f = fluorescence of the totaladded compound - fluorescence of the free compound).

observe a fluorescence enhancement, the plott ing of which reveals a coope-

rative process. Such a cooperativity has already Deen reported in the case of

the interaction of modified oligonucleotides with poly (rA) (6). No fluores-

cence change is detected when p-T.C7OPC or p-T4C,Q0PC are added to poly

(rA).

Melting curves of p-T.CgOPC-poly (rA) complexes.

Increasing the temperature of the complex formed at low temperature,

leads to i t s dissociation. Isobestic points were observed at the same wave-

length as those obtained during the t i t ra t ion of the modified poiynucleotide

with poly (rA). The hyperchromism induced by the melting is followed at 27u

nm and 310 nm and the melting curves which show a strong cooperative effect

are comparable for both wavelengths, indicating that the dissociation of the

OPC ring from the complex is correlated to the denaturation of the annealed

complex (Fig. 6). As expected from the self-association data, the increase of

the optical density at 310 nm observed for temperatures higher than 35°C is

due to the unstacking of the p-T.CgOPC molecule. The control experiments

carr ied out on p-T.C,-OPC alone allow for the corrections of this unstacking

and lead to melting curves which are quite comparable at 270 nm or 310 nm ;

in both cases, the maximum of hyperchromicity is 20 %. The Tm values are

shown in Table 2 and are about equal for p-T.CgOPC and p-T.C70PC. The above

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Q_ l

OI-0.o

0.45 -

040 -

0.35 -

O 3 0 -

40 60

TEMPERATURE [ ° c ]

Figure 6. Melt ing curves of the complex between p-T«CgOPC (A) or a-T̂ CcOPC(B) and poly (rA). a- or p-T4C5OPC concentration was 10"5M and the poly (rA)concentration was 4 .10 " 5 M nucleotide/1 corresponding to an adenine to tnymineration of 1. Absorption measurements were made at (•) 310 nm, (o) 270 nm, at(A) 310 nm only for free T4C5OPC. Curve (A) was obtained by correcting curve(•) for the temperature dependence of the free drug absorbtion measured at310 nm.

correction for unstacking assumes that a l inear approximation is adequate for

the temperature dependence of the p-T.CgOPC extinct ion. Apart from the expe-

rimental determination of this dependence ( f i g . 2) , we have used the

Marquarat non linear least squares method to f i t the melting curves, accor-

ding to Petersheim and Turner (31). This data treatment is based upon the

assumption that the cooperative part of the melting curves reflects a single-

strand to double-helix equilibrium. The derived parameters thus obtained are

in agreement with linear temperature dependences of the double-and single-

strand extinctions. The derived Tm values are very close (=0.2°C) from those

direct ly determined from the experimental melting curves, just i fy ing the

corrections for unstacking. The Tm corresponding to the complexes formed with

these compounds are much higher than the one corresponding to p-T.C mixed

with poly (rA) (Tm<O°C). These results indicate that the OPC ring strongly

stabi l ize the complex formed with poly (rA). Thermodynamic parameters for the

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Table 2. Thermodynamical parameters corresponding to the association of p or

q-T4C50PC with poly (rA).

Compounds

p-T4C5OPC

a-T4C5OPC

270

310

270

310

TmC

nm

nm

nm

nm

C)

: 16

: 16

: 24.

: 26

5

AHUcal/mole)

270

310

270

310

nm : -42.6

nm : -46.0

nm : -38.7

nm : -36.8

AS(cal/mole/°C)

270

310

270

310

nm :

nm :

nm :

nm :

-123

-130

-105.3

-98. a

These parameters have been derived at 270 nm and 310 nm. The Tm values aregiven fo r a concentrat ion of p- or g-T CKOPC equal to 10"5M i n sodium cacody-la te 10 m M pH 7 and NaCl 0.1 M. * a

binding of the modified oligonucleotides to poly (rA) can De calculated from

the p lo t of 1/Tm versus log Cm where Cm is the concentration of T.Cj-OPC at T

= Tm (31). These plots y ie ld straight lines (Fig. 7) and lead to values of AH

and AS which are given in Table 1 .

Interaction of g-T C OPC with homopolynucleotides.

The opt ical changes induced when a-T.CsOPC is mixed with increasing

concentrations of poly (rA) at tf°C appear in the same region of the spectra

as those observed with p-T-C,-OPC out no isobescic point is apparent (Fig.

3). The hypochromicity which appears in the 270 nm region of the spectra is

most l ikely due to the annealing of the C1-T4 moiety of the modified ol igo-

nucleotide to poly (rA). Such an annealing between an a-oligonucleotide and

i t s p-complementary counterpart has already been demonstrated by NMR studies

(14). As for the p-der ivat ives, one molecule of o-T.C.OPC is bound per 4

adenine residues (Fig. 4).

The hybridisation of a-T CgOPC to poly (rA) does not induce any fluores-

cence changes suggesting that the OPC ring is in a different environ-ment

than the one created by the association of p-T.CsOPC with poly (rA).

The association of a-T.C50PC with poly (rA) is temperature dependent.

Once the complex has been formed at a relat ively low temperature (8°C in

our experimental cond i t ions) , the UV spectrum of the free a-T.C.OPC can be

progressively recovered by raising the temperature. Two isobestic points

appear at 347 nm and 324 nm and the optical density changes at 270 nm and

310 nm display a cooperative ef fect. Tm values at 270 nm and 310 nm (Fig.

4) are given on Table 2 ; they are higher for the o-T.CgOPC poly (rA) complex

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m3.6

35

3.4

33

-

1 I

A

B

i

-6 -5.5 -5logCm

Figure 7. Effect of the i n i t i a l o- and p-T4C50PC concentration on the disso-ciat ion of the TÔPC-poly rA complexes.The adenine to thymine rat io was equal to 1. 0-T4C5OPC (A) and P-T4C5OPC(B) concentrations varied from 0 . 2 5 . 1 0 - 5 M and 0.30.10"5M respectively to2 . 1 0 " 5 M . Tm was determined by adsorption measurements at 270 nm (•) and 310nm (X) in pH 7 buffer containing 10 m M sodium cacodylate and 0.1 M NaCl.

than for i t s p-counterpart. The Tm value of the complex formed between a-T^

alone and poly (rA) is lower than 0°C. Again, by varying the concentration of

the components and measuring the corresponding melting temperatures (Fig. 7),

we have derived the thermodynamic parameters of this interaction.

DISCUSSION.

Our spectroscopic data show that o l igo(dT) . under an a- or p-anomeric

configuration, covalently linked to an OPC dye through a polytnethylene

tether, are able to specifically interact with the complementary sequence,

poly (rA), provided that the l inker is properly sized. The stoichiometry of

the complexes corresponds to the formation of a number of A-T base pairs

equal to that of thymines in the oligo (dT)4. When related to a-p hetero-

hybrids, this result shows that dT-rA pairing found in the p-p antiparallel

heteroduplex is also present in an a-p parallel heterohybrid ; i t also ind i -

cates that no dT-rU or no dT-rC pairing occurs in conditions where a dT-rA

pai r ing is formed in T.CgOPC complexes. This result may probably be genera-

l ized and implies that dT-rU or dT-rC pairing is not possible in any a-p DNA-

RNA heteroduplex.

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The OPC moiety of the molecule participates to the interaction as shown

by the changes observed in i ts absorption spectrum. The s tab i l i ty of the

complexes formed between the modified oligo(dT)s and poly (rA) is enhanced

as compared with the complex formed with the unmodified oligo(dT)s alone.

The features of the interaction depend markedly upon the length of the

l inker. An increase of this length from 5 to 7 carbon atoms keeps unmodified

the Tm value of the complex formed with poly (rA) but induces some modifica-

tions of absorption and fluorescence characteristics of the OPC r ing. The J10

nm spectral changes associated to the formation of the p-T.CÔPC-poly (rA)

complex are similar to those associated to the intercalative binding of OPC

alone to duplex poly (rA-rU) or to double stranded UNA (results not shown).

On the other hand, p-T.C,OPC, when bound to poly (rA), display UV absorption

spectral changes which are similar to those observed when OPC alone interacts

with single stranded poly ( rA) . In addit ion, p-T.CyOPC does not induce any

fluorescence change. These results suggest that the OPC moiety is embodied in

a different environment after binding to poly (rA) and loses i ts interca-

l a t i on a b i l i t y when i t is attached to p-T«C7 instead of p-T.Cs although each

complex displays an identical s tab i l i t y . NMR studies under way in one of our

laboratory w i l l provide more defini te stereochemical data. The lack of inter-

act ion between p-T.C.QOPC and poly (rA) was rather unexpected ; i t can be

described to a peculiar mode of self- or auto-association of the hydrophobic

T.C,UOPC molecules which is suggested by our foregoing results on the concen-

t rat ion dependence of the UV spectra and which might hinder their binding to

the anionic polyribonucleotide. The influence of the OPC riny on the s tab i l i -

zation of the complex is also demonstrated by the heat denaturation experi-

ments. A poor dependence of the Tm values on NaCl concentration suggests that

an increase of ionic strength gives r ise to two antagonist effects : a stabi-

l izat ion of the duplex between the T4 moiety of the molecule and poly (rA)

and a destabilization of the binding of the OPC moiety.

The spec t ra l changes accompanying the association of a-T-Cj-OPC to

poly (rA) as well as the absence of fluorescence enhancement are not in favor

of the occurence of a conventional intercalation process. I t has not been

possible to decide between several hypothesis which might explain the pecu-

l i a r behavior of the a-anomer compared to that of the p-anomer : inab i l i t y of

the a- and p-strands running parallel along the heterohybrid to provide space

for an intercalating molecule ; lack of fluorescence enhancement after inter-

calat ion, or preferential electrostatic attraction by the phosphate groups of

the non annealed part of the poly (rA) or steric hindrance of the OPC moiety

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linked to (1-T4 for f i t t i n g inside a potential intercalation s i te . These

problems are under active investigation in our group.

Changing the B-anomeric configuration of the oligonucleotides to the a

one, enhances the s tab i l i t y of the complex formed between a modified ol igo-

nucleotide and poly (rA). In our conditions, we cannot measure the Tm for

the a-T4 alone which l ies below 0°C ; we therefore cannot ascriDe the

enhanced s t a b i l i t y of the o-T4C50PC to a more stable structure of the a-p

duplex or to a better stabi l izat ion of the complex through the interaction

of the OPC r ing.

In conclusion, we have synthesized a new set of molecules which are

formed of an intercalating agent (oxazolopyridocarbazole) covalently linked

through a polymethylene arm, to a- or p-tetranucleotides. These molecules

exhibit a specific interaction with their poly (rA) complementary sequence.

Their binding is strongly reinforced by the energy provided by the OPC

moiety. By comparing the respective s tab i l i t ies of these molecules, i t

appears that the a-anomeric configuration imposes an additional stabi l izat ion

of the complex formed with poly {rA).

This property establishes that the rational use of p-oligonucleotides

covalently linked to UNA binder (5-10) might be extended to the correspon-

ding a-oligonucleotides, with the attract ive feature that these modified a-

oligonucleotides are resistant to isolated nucleases (17) as well as to cel -

lular extracts (unpublished results). In addition, the parallel a-B hybrids

exhibit structures which might interfere with some of the enzymatic events

associated to the regulation of genes expression, and might therefore consti-

tute potential tools for i t s control.

ACKNOWLEDGEMENTS.

One of us (C. Gaut ier) was supported by a fe l lowsh ip (BDI) from CNRS

and SAN0F1.

*To whom correspondence should be addressed

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