Identifying antisense oligonucleotide binding

Details Identifying antisense oligonucleotide binding Identifying antisense oligonucleotide binding

1999-05-26

2002-12-31

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C536S024500

Reexamination Certificate

active

06500615

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for identifying accessible sites in an RNA molecule for antisense agents and to methods for identifying antisense oligonucleotides.
BACKGROUND TO THE INVENTION
Most library strategies used for identifying oligonucleotides (ONs) that bind an RNA have relied on cleavage of the target mRNA by RNAse H as the key component of their selection process. This, however, only selects for ONs which bind their targets with normal Watson-Crick interactions. ONs that bind their target RNA can mediate cleavage by RNAse H and the resultant fragments can be isolated by high resolution gel electrophoresis. The precise sequence of the successful ON is determined by sequencing the fragments. Mishra and Toulmé (1) have developed an alternative selection procedure based on selective amplification of ODNs (Oligodeoxynucleotides) that bind their target and have demonstrated non-Watson-Crick interactions in that binding. Their protocol requires just as much sequencing as the RNAse H strategy but they sequence the ODNs and not the target RNA fragments. Furthermore most library strategies use libraries that are free in solution which has the problem of cross hybridisation of ONs within the library. This can be solved by using mulitple libraries of minimally cross hybridising subsets but this adds to the labour involved in the selection of ODNs with active antisense properties.
SUMMARY OF THE INVENTION
The present invention provides a method for identifying an antisense oligonucleotide capable of binding to a target mRNA, which comprises contacting the target mRNA with each member of an oligonucleotide library separately under hybridisation conditions, removing unhybridised material and determining which member or members hybridise; wherein the oligonucleotide library comprises a plurality of distinct nucleotide sequences of a predetermined common length, and wherein each nucleotide sequence comprises a known sequence of 4 to 8 bases and all possible combinations of the known sequence are present in the library.
Each member of the oligonucleotide library may be contacted with the target mRNA in a separate container which may be immobilised in the container. Alternatively, each member of the oligonucleotide library may be immobilised at a separate location on a hybridisation array. Preferably the length of the known sequence is from 4 to 6 bases.
Each member of the oligonucleotide library may comprise a set of nucleotide sequences. Each nucleotide sequence may have a window region comprising the known sequence and a flanking region of no more than 8 bases, wherein all possible combinations of bases in the flanking region are present in each set and the common length of the nucleotide sequences is no more than 12 bases, preferably no more than 10 bases. The nucleotide sequences may be made from DNA analogues.
In one arrangement, the step of determining which member or members hybridises comprises determining the member or members which hybridise more rapidly.
The sequence of each member which hybridises may be compared with sequence information relating to the target mRNA so as to identify in the target mRNA one or more antisense binding sites.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention is aimed at the problem of identifying accessible sites within an mRNA but the object of this technique is to find accessible sites in a system that is applicable to any RNA molecule, that requires no sequencing and is not biased by the use of RNAse H. The emphasis of this patent is on the use of libraries of short oligonucleotide (ON) probes, preferably 4-mers. This invention provides a method for determining the accessible sites of an RNA to short ON probes by following the hybridisation reactions of each of the probes in the library with the target. Probes are spatially isolated and in the reactions, and either the probe or the target RNA is immobilised.
The rationale behind using probes with windows as short as 4mers is the assumption that most occurrences of a given 4-mer will be sequestered in secondary or tertiary structure. The data gained from following each individual hybridisation is interpreted in terms of the primary sequence:—hybridisation of several 4 mers to an accessible region will be marked by clustering of overlapping 4-mers in the primary sequence of the RNA. Ambiguities should be resolvable in part by analysis of the binding kinetics, so even if a 4-mer occurs more than once, it should still be possible in most cases to identify the most accessible regions in the RNA. The importance of using short probes is the ability to calibrate and normalise the hybridisation behaviour of all members of the library, since the library is not so large that this is impractical. A 4-mer library has 256 members, which is a modest number and it is realistic to test such a library against a number of model molecules to calibrate the system. Calibration is important as most 4-mers have different binding energies for a given degree of accessibility. In order to identify the regions of a molecule that are accessible on the basis of hybridisation kinetics requires that one be able to compare normalised kinetic data that takes into account that discrete overlapping 4-mers will bind the same accessible site with different kinetics. Small arrays of probes are cheaper to construct and manipulate when spatial isolation of probes is desired. Furthermore short probes are much less likely to have any secondary or tertiary structure themselves which would complicate the analysis of the hybridisation reactions.
Hybridisation times can be varied to derive kinetic data about each hybridisation reaction. The amount of probe ON hybridised after a given time will give quantitative data about the binding affinity of that probe for its target. By varying the hybridisation duration, a time course for the hybridisation reaction of each probe can be derived. The accessibility of any region of the structure will be inversely related to the time its complementary probe will take to bind hence detailed information about secondary structure can be acquired by this approach. In combination with a Scintillation Proximity Assay (Amersham), discussed below, the system is still more effective in that no washing is needed, so shorter probes can be used and the hybridisation reaction of each probe can be followed in real time.
Molecular Probes of RNA Structure
Spatial isolation and immobilisation of probes avoids cross-hybridisation problems associated with the use of oligonucleotide libraries free in solution simplifying interpretation of results. This process does not necessarily determine the most effective cut sites for RNAse H but provides detailed structural information about the target RNA to allow rational design of targetted molecules which can be then be developed into effective antisense agents, which will not necessarily operate on the basis of RNAse H mediated RNA degradation.
Advantages Over Previous Library Strategies
This invention uses random libraries of ONs since, in a random library of ONs of a given length, every possible sequence of that length is represented, hence every sub-sequence of that length that comprises a given target RNA is also represented. This means that a random library will be applicable to any RNA and will contain all the target subsequences within it. Since ONs are monitored independently, this means that this approach entails the targeted libraries discussed in a previous patent PCT/GB96/02275, and gives detailed kinetic behaviour about them. Explicit data concerning accessibility of sub-regions of the RNA to normal Watson-Crick interactions can thus be derived by this system.
Non-Watson-Crick interactions might well be less common than the normal complementary interactions but will also be represented in a system like this. One would expect interactions with regions that are partially complementary to the target RNA. If Watson-Crick interactions are the only potential interactions that are possible, such partial hybridisation would be ex

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