Enzymatic methods of preparing polymers from nucleotide...

Details Enzymatic methods of preparing polymers from nucleotide... Enzymatic methods of preparing polymers from nucleotide...

1999-06-22

2001-12-11

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C536S022100, C536S025300, C536S025320

Reexamination Certificate

active

06329177

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit from German application serial number 198 27 728.8, filed Jun. 22, 1998, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention is directed to the preparation of natural and/or modified polymers having a defined sequence consisting of nucleotide and/or non-nucleotide monomers in an enzymatic manner.
TECHNICAL BACKGROUND
Synthetic oligonucleotides and polynucleotides having a defined sequence are of use in diagnostics and as therapeutics, for example as antisense oligonucleotides, ribozymes or aptamers.
Oligonucleotides may be prepared using chemical or enzymatic methods. The enzymatic method as an alternative of the conventional chemical process is gaining more and more importance. Known enzymatic techniques proceed in 5′→3′ direction.
For the preparation of oligonucleotides in an enzymatic manner, the following chain elongating enzymes may be used:
a terminal transferase,
a polynucleotide phosphorylase,
a RNA ligase, or
a DNA polymerase (EP 0 491 739 B1).
Terminal transferase, i.e. terminal deoxynucleotidyl transferase (EC 2.7.7.31), catalyses the addition of deoxynucleotide triphosphates to the 3′-OH terminal of single or double stranded DNA in a template-independent reaction.
Polynucleotide phosphorylase (EC 2.7.7.8) catalyses a number of reactions, among them the 5′→3′-polymerization which involves ribonucleoside 5′-diphosphates.
RNA ligase (EC 6.5.1.3) catalyses a covalent linkage between a 5′-phosphate donor and a 3′-hydroxy acceptor in the presence of ATP.
It is obvious from the reaction mechanism of RNA ligase that an ATP dependent and an ATP independent step can be distinguished in the course the reaction steps. First, the enzyme binds a molecule of ATP at the ATP binding site, and in a first reaction step AMP is covalently linked to the enzyme with simultaneous release of PP
i
. The donor is bound by its enzyme binding site via the 5′-terminal nucleoside and the two neighbouring phosphate groups. Then, in the second reaction step, donor activation may occur by transfer of the enzyme-bound AMP to the 5′-terminal phosphate group of the donor while the phosphoanhydride bond is formed. Donor activation takes place in an ATP-dependent step. In the third step of the reaction the internucleosidic bond between acceptor and donor is formed while AMP is released. Ligation of the activated donor A-5′-pp5′-N to the acceptor is ATP independent. Both reaction steps may also proceed in a manner independent of each other.
The enzymatic synthesis of oligoribonucleotides in 5′→3′-direction is known. In the variant of the synthesis according to Hyman /1/ the acceptor and an adenylated nucleoside 3′,5′-bisposphate with a phosphate protecting group in 3′-position are presented to RNA ligase as the activated donor. This reaction is illustrated by the scheme shown in FIG.
1
.
The abbreviations in
FIG. 1
have the following meanings:
AppNp an activated donor, a nucleotide having a phosphate protecting group in 3′-position,
pNp a nucleotide having a phosphate protecting group in 3′-position,
pN a nucleotide without protecting group in 3′-position.
The method of Hyman is well suited for the synthesis of natural and modified oligoribonucleotides.
For the synthesis of oligodeoxyribonucleotides, Hyman proposes
to synthesize a complementary oligoribonucleotide according to his method and
to convert the oligoribonucleotide into a oligodeoxyribonucleotide using reverse transcriptase /1/2/.
Other methods describe the amplification of a template using DNA polymerase I /3/4/.
These known methods bear several disadvantages.
The first disadvantage is that no oligonucleotides with alternating sequences, such as with natural phosphodiester or thiophosphate bonds or with natural ribose residues or 2′-alkyl substituted ribose residues, may be prepared but only a uniform monomer type may be polymerized.
The second disadvantage is that not the extraordinary broad substrate specificity of RNA ligase for the donor can be used to synthesize a modified product, insofar as the same modification is desired in all monomers, but instead the very limited substrate specificity of reverse transcriptase must be employed.
At the moment, RNA ligase may only be used in the synthesis of natural and modified oligoribonucleotides. The reason for this is the very narrow specificity of RNA ligase for an oligoribonucleotide being the acceptor.
If the newly synthesized growing product is the acceptor, one or two deoxyribonucleotides may be coupled, then the method is no longer practical since RNA ligase is unable to incorporate oligodeoxyribonucleotides as acceptors in the polymerization reaction in 5′→3′ direction.
Known studies concerning the enzymatic synthesis of oligodeoxyribonucleotides in 5′→3′ direction are limited to complex methods which are impracticable on a technical scale /5/6/.
SUMMARY OF THE INVENTION
The present invention is based on the object of providing novel enzymatic methods for general use using a RNA ligase for the preparation of different polymers from nucleotide and/or non-nucleotide monomers, in particular for the preparation of natural and/or base modified and/or sugar-modified and/or phosphate group-modified oligodeoxyribonucleotides and/or oligoribonucleotides and/or non-nucleotide polymers in 3′→5′ direction having a defined monomer sequence. The polymers which may be prepared according to the invention also comprise oligonucleotides and polynucleotides in their most general form. It is another object to provide carrier molecules to carry out the methods.
The solution of these objects will become obvious from the following specification and the claims.


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patent: 5750341 (1998-05-01), Macevicz
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McConnell, T.S. et al., “Guanosine Binding to the Tetrahymena Ribozyme: Thermodynamic Coupling with Oligonucleotide Binding”,Proc. Natl. Acad. Sci. U.S.A., vol. 90, No. 18 (1993), pp. 8362-8366.
Shevchenko, N.M. et al., “Synthesis of Modified Triuridylates by the H-Phosphonate Method”,Bioorg. Khim., vol. 14, No. 7, (1988), pp. 976-978 (English Abstract Only).
Holy, A. et al., “Effect of 5′-Substitution on the Template Activity of Oligonucleotides for the Binding of Valine and Alanine tRNA to Ribosomes”,Biochim. Biophys. Acta., vol. 217, No. 2, (1970), pp. 332-345.
Kleinwachter V. et al., “Luminescence Properties of Dinucleoside Phosphates, Some Oligonucleotides, and Polynucleotides”,Collect. Czech. Chem. Commun., vol. 37, No. 10 (1972), pp. 3433-3446.
Vratskikh, L.V. et al., “Solid-Phase Synthesis of Oligoribonucleotides Using T4 RNA Ligase and T4 Polynucleotide Kinase”,Biochimie, vol. 77, No. 4 (1995) pp. 227-232.

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