Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-12-23
2001-02-13
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025320, C501S033000, C560S155000, C428S402000, C546S025000, C546S102000, C548S304100, C549S223000, C549S227000
Reexamination Certificate
active
06187913
ABSTRACT:
The present invention relates to covalently crosslinked duplex oligonucleotides, that is to say DNA, RNA or mixed DNA/RNA duplexes the two complementary strands of which are crosslinked covalently.
More specifically, the present invention relates to a single-stranded oligonucleotide possessing intra-oligonucleotide covalent link(s), that is to say a single-stranded oligonucleotide which contains a covalent link between the two sites of the oligonucleotide, or several covalent links between a number, a multiple of two, of sites of the oligonucleotide. In particular, the oligonucleotide can be of the “hairpin” type, that is to say containing two paired fragments of the same strand separated by a non-self-paired loop, the covalent link being located in said paired fragments.
The present invention also relates to a double-stranded oligonucleotide possessing inter-oligonucleotide covalent link(s), that is to say a double-stranded oligonucleotide in which the two paired strands are linked to one another via covalent links between one or more sites of each of said two strands.
Oligonucleotides of this type have been described in Patent Application PCT WO93/18052. They are useful in a therapeutic application, in particular in the so-called “sense” strategy where they are used for their property of hybridizing with factors involved in the transcription of genes involved in pathologies. Sense agents of this type have also been described in Patent Application PCT WO92/19732.
Crosslinked oligonucleotides are also useful as research reagents for purposes of elucidation of the mechanism of DNA-protein interaction. In particular when this interaction is associated with a disturbance of the structure of the double helix (RNA polymerase, methylase, uracylglycosilase and other repair enzymes).
The practical use of crosslinked duplexes enables the level of gene expression in different tissues to be studied in in vitro systems (when specific factors take part in this mechanism), and constitutes an effective means for inhibiting transcription and modulating it.
These duplexes have the especially advantageous property of being more resistant to endonucleases and exonucleases than duplex oligonucleotides without covalent bonding.
An objective of the present invention is to provide oligonucleotides possessing improved covalent crosslinking, that is to say the covalent link must be sufficiently flexible to comply with the helical structure of the duplex or of other secondary and tertiary structures of the DNA.
Another objective of the present invention is to provide a simplified method for preparing these covalently crosslinked oligonucleotides.
A further objective of the present invention is to obtain a covalent link which can be cut without disrupting the hybridization of the covalently linked oligonucleotides or portions of oligonucleotides.
In the document WO93/18052, covalently cross-linked duplexes have been described in which the covalent link results from the reaction of an aldehyde group, more specifically a dialdehyde group, and an amine group. This reaction yields a Schiff's base, followed by a reduction with a borohydride which results in a covalent hydrazide linkage. A reaction is also described between an aldehyde and hydroxyl groups, which results in an acetal linkage.
The present invention consists in using a covalent link of the amide type
between a primary amine group and a carboxyl group. This type of linkage makes it possible to achieve the objectives set by the present invention.
Although this type of amide link is advantageous since its chemistry is much simpler, a single condensation reaction in the presence of a condensing reagent of the carbodiimide type is necessary, it was not described in the document WO93/18052 for the following reason. This type of amide link, when it involves two nucleic acids, more specifically two oligonucleotides, must be produced in an acid medium at a pH of approximately 6.5. Now, the pKa of an aliphatic primary amine group is of the order of 11. Under the reaction pH conditions of the order of 6.5, the aliphatic primary amine group is hence predominantly in protonated form and the nucleophilic form of the amine is virtually nonexistent, so that the reaction cannot take place or, at all events, does so in very low yield.
However, according to the present invention, advantage has been taken of the fact that an amine group situated at the 2′ position of a 2′-deoxynucleotide possesses a markedly lower pKa, namely of the order of 7.4, which permits coupling of this amine with a carboxyl group under pH conditions compatible with the coupling of two oligonucleotides. As a result of the reaction pH level, it is maintained to a greater extent in free, unprotonated form, thus remaining nucleophilic.
An essential feature of the present invention is hence to use a modified nucleotide carrying an NH
2
group at the 2′ position of a 2′-deoxynucleotide, coupled to another modified nucleotide or modified nucleotide analog carrying an aliphatic group having a carboxyl group at its end.
More specifically, the subject of the present invention is a double-stranded or single-stranded oligonucleotide containing one or more inter- or intra-oligonucleotide covalent crosslink(s), respectively, characterized in that the or each covalent link consists of an amide linkage between a primary amine group of one strand and a carboxyl group of the other strand or of the same strand, respectively, said primary amine group being substituted directly at the 2′ position of the saccharide ring of a nucleotide of one strand, and said carboxyl group being carried by an aliphatic spacer group substituted on a nucleotide or a nucleotide analog of the other strand or of the same strand, respectively.
“Nucleotide analog” is understood to mean a non-nucleotide insert which contains, like the natural nucleotides, a linear motif of 3 carbon atoms between the two inter-nucleotide links, so that said motif has as its skeleton:
This non-nucleotide insert makes it possible to adhere to the natural inter-nucleotide distance. Said “nucleotide analog” takes the form of a monovalent residue or of a divalent residue if it replaces a terminal nucleotide or a nonterminal nucleotide, respectively, in the oligonucleotide.
The aliphatic group included in the covalent link serves as a spacer group, since its size must be such that the covalent link between the two oligonucleotides in question comprises a linear chain whose skeleton contains from 5 to 25 aligned atoms, so that the distance between the two strands on the one hand and the spatial conformation of the double helix on the other hand are adhered to and permit good hybridization between the two strands.
In one embodiment, said carboxyl group is carried by a nucleotide analog consisting of a non-nucleotide insert of the 1,3-propanediol type substituted at the 2 position with an aliphatic group (R) carrying said carboxyl group at its end.
In an other embodiment, said carboxyl group is carried by an aliphatic group substituted at the 2′ position of a 2′-deoxynucleotide.
However, it is preferable to use a nucleotide analog, since the reactions are easier to carry out inasmuch as there is no need to protect the nucleotide in the various other functional groups of the saccharide ring and/or of the purine or pyrimidine base.
The nucleotide(s) and nucleotide analog, where appropriate, involved in an inter- or intramolecular covalent link can be in a pairing position, that is to say face to face on each strand of the paired fragments.
However, depending on the size of said aliphatic group, it can be preferable in some cases for the nucleotide(s) and nucleotide analog involved in a covalent link not to be in a pairing position, and to be offset by one or more nucleotides relative to the pairing position. Thus, suitably, the nucleotide(s) and nucleotide analog involved in a covalent link are offset by 1 to 5 nucleotides relative to the pairing position.
Advantageously, said aliphatic group contains a linear cha
Blumenfeld Marta
Merenkova Irena
GENSET
Pennie & Edmonds LLP
Wilson James O.
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