DD273445A1 - PROCESS FOR PREPARING COVALENT CONJUGATES FROM OLIGODESOXYNUCLEOTIDES AND POLYAMINOSAURES OR PROTEINS - Google Patents

Abstract

The invention relates to a process for the preparation of covalent conjugates of oligodeoxynucleotides and polyamine acids or proteins. Thus, oligodeoxynucleotides which can be up to 70 nucleotides long in any desired sequence are linked to polyamine acids or proteins in a simple manner in good yield. The resulting conjugates of general formula (III) oligodeoxynucleotide-3OO oligodeoxynucleotide-3OPO (CH2) nNHCO (CH2) 2CONHR (III) oligodeoxynucleotide-3OPOn2-6; RPolyaminosaeure, protein inhibitors in cells by hybridization with cell-own or viral nucleic acids, the expression of the corresponding genes specific. Field of application of the invention are molecular biology, medicine and microbiology.

Description

in which an oligodeoxynucleotide is present with up to 70 nucleotides in length and η = 2-6, with polyamino acids or proteins of the general formula II

R-NH ₂ (H)

under the action of a carboxyl group activating reagent to conjugates of the general formula III

Il

Ol i qncJb? O>: ym.u: 1 eot i cJ-3 '- (JPO - <CI Iv.) ,, - NH-CO- <CH _M ) _si -CO -NH- R ,,,.

, f

in which R represents a polyamino acid or a protein.

2. The method according to claim 1, characterized in that is used as the carboxyl group activating reagent i-ethyl-S-IS-dimethylaminopropyD-carbodiimide.

Application blot of the invention

The invention relates to a process for the preparation of covalent conjugates of oligodeoxynucleotides and polyamino acids or proteins and the use of the conjugates for the specific inhibition of the expression of the genes complementary to the oligonucleotides in cells. Field of application of the invention are molecular biology, medicine and microbiology.

Characteristic of the known state of the art

Derivatization of oligodeoxynucleotides following synthesis at the 5 'end by condensation with appropriate reagents has been described several times without the derivatives being used to couple the oligodeoxynucleotides to proteins or polyamino acids (S.Agrawal, C.Christodoulou, MJ Gait, Nucleic Acids Res. 14 [1986] 6227, T.Horn, MS Urdes, DNA, J. Molec. Biol. 5 (1986) 241; L. Wächter, J.-A. Jablonski, KL Ramachandran, Nucleic Acids Res. 14 [1986 ] 7985;

JM Kremsky, JL Wooters, JP Dougherty, RE Meyers, M. Collins, EL Brown, Nucleic Acids Res. 15 (1987) 2891, BA Conolly, Nucleic Acids Res. 15 [1987] 3131, F. Soela, K.Kaiser, Nucleic Acids Res. 15 [1987] 3113; R Bischoff, JM Cuouil, FERegnier, Analyt. Biochem., 164 [1987] 336).

Short peptides can be used as spacers in the oligodeoxynucleotide synthesis and allow the linking of the 3'-terminus of the oligodeoxynucleotide with the N-terminus of a peptide that must not interfere with the oligonucleotide synthesis and usually consists only of the same amino acid without a functional side chain (J. Haralambidis , L. Duncan, GWTregear, Tetrahedron Lett. 28 [1L87] 5199).

For the substitution of the 3 'end of the oligodeoxynucleotide chain, two methods are known: By enzymatic condensation with a Ribonuklaisiddiphosphat using T4 RNA ligase, hydrolysis d6i 3'-phosphate with alkaline phosphatase and oxidation with periodate an aldehyde is obtained with Amino groups of the polylysine can be condensed (M. Lemaitre, B. Bayard, B. Lebleu, Proc. Nat. Acad., 84 (1986) 648).

By starting deoxyribonucleotide synthesis with a derivative d.is deoxycytidine, which is obtained by treatment with ethylenediamine and bisulfite and carries an aliphatic amino group, a deoxyribonucleotide is obtained, which via the 3'-amino group z. B. may be coupled to alkaline phosphatase (P.Li, P.P. Mendon, D.C. Skingle, J.A. Lanser, R.H. Symons, Nucleic Acids Res. 15 [1987] 5275). The boids last described methods are multi-level and expensive to carry out. In the second case, a sequence is obtained which immei at the 3 'end carries a cytidine, which means in most cases a "mismatch." Coupling of 3'-carboxyl-modified oligodeoxynucleotides is not yet known.

That by Lemaitre et al. oligodeoxynucleotide-polylysine conjugate (5'-TGTCATTAGi ι iAC-3'-poly-L-lysine) obtained by the elaborate synthesis method was used for the specific inhibition of the replication of the Vesicular stomatitis virus. Only information is given about a 15-hour inhibition period, in which the virus titer decreases significantly and the synthesis of two virus proteins is reduced. The further course of the inhibition is not described. A particular disadvantage of the process consists in the complicated synthesis route, which results in an overall yield of only 25-30%.

Destination of the Eiflndung

The object of the invention is to produce in a simple manner covalent conjugates between 3'-carboxyl-modified oligodeoxynucleotides of any sequence and polyamino acids or proteins which are said to be suitable for the specific inhibition of the expression of cellular or viral genes.

Explanation of the essence of the invention

According to the invention, 3'-carboxyl-modified oligodeoxynucleotides of the general formula I

U Il

Ol i qoiJtt ^ o :: ynuk I t> oh i d ~ ^ ' -DPU-- <Π! Ι ·. _; ) "-NH-CO- <CH _V. ):. _; "COOH ₍ |)

(I

in which an oligodeoxynucleotide is present with up to 70 nucleotides in length and η = 2-6, with polyamino acids or proteins of the general formula II

_{(R-NH 2} (II)

where R corresponds to the polyamino acid or protein radical,

by the action of a carboxyl group activating reagent by the action of a carboxyl group activating reagent such. B. i-ethyl-S-is-dimethylaminopropyD carbodiimide hydrochloride at a pH of 4-6 to a conjugate of the general formula! ! I!

O Il

Ol iqodt; - ;; o>: ynuk I eoti d -3 '- DPD-- (CW _x ) ,, - MH - CO- (CH _H ) -. · CO-NH-R

I (IH)

is condensed. By the method according to the invention it is possible to easily condense oligonucleotides via their 3'-Eode with amino group-containing molecules such as polyamino acids or proteins and to isolate the conjugates of general formula III in 80-90% yield.

The oligodeoxynucleotides A-F used to inhibit Gro-T antigen expression in COS cells in the form of poly-L-lysine conjugates are further specified in FIG. When treating the cells with the conjugates, up to 74% inhibition of the synthesis of the T antigen is observed (Table 1). If CV-1 cells infected with SV-40 virus are treated once or several times with the conjugates, the number of cells expressing T antigen is surprisingly reduced by 85% on untreated cells on the 7th day after infection (Tab .2). A treatment on the 1st, 2nd and 3rd day after infection has proven to be particularly effective. In contrast, non-complementary oligodeoxynucleotides or their conjugates as well as the complementary oligodeoxynucleotides themselves or their non-covalent complexes with poly-L-lysine did not show any inhibiting effect. It can be concluded from this that only covalent complexes between poly-L-isine and the oligodeoxynucleotides complementary to the gene sequence to be inhibited specifically and effectively inhibit the expression of the gene.

The production of the 3'-carboxyl-substituted oligodeoxynucleotides is carried out by the phosphoramidite method, using a carrier-bound spacer of the following structure

-CO-NH-ICHjln-O-COHCHjh-CO-NH-ICHjJn-OH.

After the synthesis of the oligodeoxynucleotides, the spacer is replaced by 48 hrs. Reaction with triethylamine-water mixture at 37 ⁰ C hydrolyzed, thereby releasing the Karboxylgruppt and replaced the oligodeoxynucleotide from the carrier. The removal of the other protective groups is then carried out by 4 hours. Heating in concentrated, aqueous ammonia at 56 ⁰ C.

Ausführungsbelsplel

Preparation of the Carboxyl-Substituted Oligodeoxynucleotide Having the Nuclide Structure B'-CCTCACTACTTCTGGAATAGC-a 'at η = 2

According to the known phosphoramidite method, the oligodeoxynucleotide is synthesized by sequential condensation of the corresponding phosphoramidites on the above-mentioned spacer, which after hydrolysis of the spacer carries a carboxyl group corresponding to the general formula I.

Preparation of the conjugate

0.1 μΜοΙ oligodeoxynucleotide of general formula I having a nucleotide structure B'-CCTCACTACTTCTGGAATAGC-a 'is suspended in 0.2 ml of buffer containing 0.1 M morpholinoethanesulfonic acid, pH 5.0, and 2.5 M sodium chloride, with 1 μΜοΙ 1 -Ethyl-3- (3-dimethylaminopropyD-carbodiimide hydrochloride (EDC-HCl) at 20 ° C for the reaction, after 5 minutes, 0.1 μΜοΙ poly-L-lysine of average molecular weight 0000 was added and after 5 minutes again 1 After 16 hours at 20 ° C., the reaction mixture is filtered through a Sephadex G 25 column and the conjugate is isolated as the first fraction in an 80-90% yield.

The condensation between the oligodeoxynucleotide and poly-L-lysine is detected spectrophotometrically and by protein determination. The conjugate has an average composition of 1 mole of oligodeoxynucleotide per mole of poly-L-lysine.

Inhibition of expression of the T-antigen gene of SV40 virus in COS cells

For culture of COS cells in MEM medium, the conjugate prepared according to the above protocol is added in concentrations between 5 to 20 μΜ and left for 20 hours. During this time, the protein newly synthesized in the cells is labeled with ³⁵ S-methionine and the resulting. T-antigen precipitated after digestion of the cells with a specific antiserum. The amount of T-antigen is determined by polyacrylamide electrophoresis and autoradiography. The expression of T-antigen is reduced by 75% by addition of the conjugate, without the synthesis of cellular proteins is reduced.

Inhibition of the proliferation of SV40 viruses in CV-1 cells

For culture of CV-1 cells in MEM medium, the mentioned conjugate is added at a concentration of 1 μΜ either 4 hours prior to infection with SV40 virus-containing medium or simultaneously or 4 hours later. After 24 hours, the medium is changed and conjugate is added again to some of the samples. This addition can be repeated on the third day. On the 4th day, the cells are trypsinized and cultivated on coverslips. On the 6th and 7th day, samples are taken, fixed, treated with anti-T-serum and stained with FITC-labeled anti-hamster serum. By evaluation under the fluorescence microscope, the proportion of fluorescent nuclei is determined. The proportion of these cells expressing T antigen is increased by treatment with conjugate, especially with multiple doses in 24 hours. Distance reduced by up to 90%. There is no difference between the samples treated before, after or at the same time as the infection within one day.

Schematic representation of the 5 'terrestrial part of the T antigen mRNA and the complementary oligonucleotides

cap AUB

I (Γϊ21 "-7) I <5146)

A 21 - m sir O 25   -int? r c 20 IiIfH D 4 3 --tner e ^r j ^r "> - in goal F '"ι "".< i.  -me r

5211 · · 5191 5211 - 5187 5218 - 5191 5211 - 516? 5188 - 5167 5164 - 5143

Table 1

HOMAGE of T-Exprosslon of T-antigen in COS cells by antisense ollodeoxynucleoside, which were covalently conjugated to poly-L-lysines ¹ '.

Oligodeoxynucleotide ^b) conjugated with poly-L-lysine (molecular weight) Inhibition of expression A 3700 6000 52% ^cl 74% ^cl D 3700 6000 no inhibition ^dl 25% F 6000 C0% ^cl

a Inhibition is based on untreated controls and represents the mean of at least 3 trials.

b The concentration of the ollodeoxynucleotide in the form of the conugate is ΒΟμηι per ml of medium.

c standard deviation 10%.

d There is no comment, or the inhibition l'.t less than 10%.

Table 2

Inhibition of T antigen expression in SV40 virus-infected-1 CV-1 cells by antisense oligodeoxynucleotides covalently conjugated to poly-L-lysines "

Oligodeoxynucleotide ^bl conjugated with poly-L-lysine (molecular weight) Treatment of CV-1 cells with conjunct on the 1st day on the 1st and 2nd day 6th day / 7th Day on the 1st, 2nd and 3rd day Analysis: 6.Teg / 7.Day 82% / 75% 79% / 63% 6th day / 7th Day A 3700 6000 51% / 32% 55% / 41% 75% / 70% 85% / 80% 86% / 72% B 3700 52% / 49% 88% / 72% 62% / 73% C 6000 85% / 69% 22% / 25% 80% / 85% D 3700 6000 no inhibition ⁰ '21% / 12% n.d. 26% / 32% F 6000 35% / 41% n.d.

a The inhibition is related to non-controlled controls and represents the mean of at least 3 experiments. b The concentration of oligodeoxynucleotide in the form of the conjugate is 10 μg per ml medium, c There is no inhibition or the inhibition is less than 10%.

Claims (1)

1. A process for the preparation of covalent conjugates of oligodeoxynucleotides and polyamino acids odor proteins, characterized in that 3'-carboxyl-substituted oligodeoxynucleotides of the general formula I. ο
Il Ol iciodhioxynuklcHil: Ui -, i'-O-O- (CII-.),., · NH-CO- <CD · · · · ·; COOM, ₍ .