Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-13
2002-03-12
Houtteman, Scott W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024500
Reexamination Certificate
active
06355418
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an oligonucleotide library, more particularly a chimaeric oligonucleotide library, and uses thereof in the identification of antisense binding sites in target mRNA and in providing potential therapeutic agents.
BACKGROUND TO THE INVENTION
Antisense oligonucleotides are single-stranded nucleic acids which are complementary to the coding or “sense” strand of genetic material. An antisense oligonucleotide is therefore also complementary to the mRNA produced from the genetic material. If antisense DNA or RNA is present in a cell with the mRNA, hybridisation takes place to form a duplex thereby preventing translation of the mRNA by ribosomes to make a protein. Thus, antisense RNA can be used to block the expression of genes that make proteins.
The antisense approach to the inhibition of gene expression, though conceptually straightforward, presents technologically demanding challenges. A variety of approaches have been taken by various academic groups and biotechnology companies. Oligonucleotides have been made with sugar modifications, such as 2′-O allyl ribonucleotides, and with backbone modifications in the phosphate group, such as phosphorothioate deoxyribonucleotides. However, production of these individual oligonucleotides for application as antisense therapeutics, reagents or tools for drug target validation has been hampered because methods of identifying potentially efficacious antisense compounds against a given target mRNA are extremely difficult. Even with an mRNA of known sequence, it is often impossible to predict what sub-sequences in the target mRNA might be available for antisense binding because of the three-dimensional structure of the mRNA and the association of RNA with proteins.
In an alternative approach to the use of chemically modified oligonucleotides, Lieber and Strauss (ref 22) report the use of a ribozyme expression library for the purpose of selecting cleavage sites in target RNAs. The ribozyme approach suffers from the disadvantage that it requires cleavage sites containing GUC or CUC and thus is not generally applicable to all possible cleavage sites. In addition, cleavage efficiency is relatively low, and chemical synthesis of ribozyme libraries is difficult.
SUMMARY OF THE INVENTION
According to one aspect, the present invention provides a chimaeric oligonucleotide library for use in identifying an antisense binding site in a target mRNA, which comprises a plurality of distinct chimaeric oligonucleotides capable of hybridizing to mRNA to form a duplex, the nucleotide sequences of which each have a common length of 7 to 20 bases and are generated randomly or generated from information characterising the sequence of the target mRNA, wherein substantially all the nucleotide sequences of said common length which are present as sub-sequences in the target mRNA are present in the library, and wherein each nucleotide sequence comprises:
a) a recognition region comprising a sequence of nucleotides which is recognisable by a duplex-cutting RNAase when hybridized to the mRNA, and
b) a flanking region comprising a sequence of chemically-modified nucleotides which binds to the mRNA sufficiently tightly to stabilise the duplex for cutting of the mRNA in the duplex by the duplex-cutting RNAase, wherein the nucleotides constituting the flanking region are different from those constituting the recognition region; and wherein each oligonucleotide is protected against exonuclease attack.
The mRNA may be from human or other mammalian origin or from invertebrates. The term mRNA as used herein also encompasses the corresponding RNA from plants, viruses and bacteria.
Chimaeric Oligonucleotide
Each chimaeric oligonucleotide forming the library may be made synthetically using any commonly-available oligonucleotide sequence synthesizer. The exact length of the nucleotide sequence will reflect a balance between achieving the necessary specificity and keeping the length to a minimum to minimise cost. Preferably, the nucleotide sequence has a length in the range 10 to 20, more preferably 14 to 17 bases, yet more preferably around 15 bases.
The oligonucleotide is preferably protected against-nuclease attack so as to minimise degradation in the cell and increase its stability. This is particularly important in the design of an antisense compound for therapeutic use. Protection against exonuclease attack may be achieved by protecting one or preferably both ends of the oligonucleotide, for example by reverse T. or any other well-known method. Selection of the nucleotides constituting the recognition and flanking regions may also contribute to stability against nuclease because some nucleotides are more nuclease-resistant than others.
Preferably, each chimaeric oligonucleotide comprises two flanking regions, one on either side of the recognition region. In this way, the recognition region may be thought of as a “window” flanked by the two flanking regions so as to form with the mRNA a substrate for the duplex-cutting RNAase. In a preferred embodiment, each of the flanking regions is protected against exonuclease attack, preferably by reverse T. A preferred duplex-cutting RNAase is RNAase H, advantageously endogenous RNAase H (Ref 23).
The nucleotides constituting the recognition region are either modified or unmodified nucleotides and are preferably deoxyribonucleotides or phosphorothioate deoxyribonucleotides (see
FIG. 4
c
). These nucleotides are recognisable by RNAase H when hybridized to mRNA. Typically, the recognition region comprises at least four nucleotides, preferably 5 to 10 nucleotides. In a particularly preferred embodiment, the recognition region comprises five nucleotides.
The nucleotides constituting the flanking region are chemically modified so as to increase the binding constant of the oligonucleotide for hybridization to the target mRNA and preferably to increase stability of the oligonucleotide in vivo. For a particular antisense oligonucleotide, the efficiency of hybridization to mRNA is a function of concentration. Thus, to improve hybridization as a given concentration, the stability of the hybrid duplex must be increased. A number of chemical modifications can be introduced into the oligonucleotide for this purpose and these fall into three broad classes (see also
FIG. 1
, regions 1, 2 and 3):
Sugar Modifications
Various modifications to the 2′ position in the sugar moiety may be made. For example, both 2′-O methyl oligoribonucleotides and 2′-O allyl oligoribonucleotides may be useful (see references 1 and 2 and see also
FIG. 2
a
and
b
). These analogues do not form hybrid duplexes with RNA which are substrates for RNAase H. In a particularly preferred embodiment of the present invention, two flanking regions, each having four or five of one of the modified sugar-containing oligoribonucleotides, flank a window region of four or five normal deoxyribonucleotides. The window region will thereby allow cleavage of the mRNA and the sugar-modified flanking regions increase the binding of the chimaeric oligonucleotide to the mRNA. Other 2′ sugar modifications which may be used include F-substituted and NH
2
-substituted oligoribonucleotides (see
FIGS. 2
c
and
2
d
and references 3 and 4).
Base Modifications
The chemically-modified nucleotides constituting the flanking region may be modified in the base moiety. The propyne analogues of dT and dC, 5-propynyl deoxyuridine (see
FIG. 3
a
) and 5-propynyl deoxycytidine (see
FIG. 3
b
), both increase the duplex hybridization temperature and stabilize the duplex. This stabilization may be due to increased strength of hydrogen bonding to each Watson-Crick partner or increased base stacking (or both). 2-amino adenine is an analogue of dA (see
FIG. 3
c
) and also increases the stability of the duplex. This may be due to the formation of a third hydrogen bond with thymine. The 2-amino adenine-thymine base pair is intermediate in stability between a G.C and a A.T base pair.
Phosphate Modifications
The chemically-modified nucleotid
Burns Doane Swecker & Mathis L.L.P.
Houtteman Scott W.
Xzillion GmbH & Co. KG
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