Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Patent
1996-10-02
2000-06-27
Sisson, Bradley L.
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
435 6, 435 911, 536 231, 536 243, G01N 3300, C12Q 168, C12P 1934, C07H 2104
Patent
active
060805854
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
Interactions between molecules form the basis of most biological processes; understanding these interactions is important for the development of applications in basic research and medicine. For example, many drugs act by binding to specific receptor molecules. The task of finding ligands that bind to a given target with high specificity and affinity is often difficult and though the introduction of combinatorial chemistries will make this task easier, it is likely that single ligands for a single biological target may not be effective enough for some purposes; for example, where the aim is to block completely a specific process.
The present invention describes novel ways of discovering combinations of ligands which act together to produce more specific and stronger interactions than can be achieved by a single ligand.
2. Description of Related Art
There are two distinct ways in which ligands could act cooperatively: such as proteins and RNA, are held in their active conformation by intramolecular interactions based on weak forces. Binding one ligand to the molecule partially opens its structure, and, as we will show, may expose it to other ligands which cannot bind in the absence of the first ligand. These additional ligands will reinforce the attenuation of the target molecule. one dependent on a different macromolecule; for example, most of the pathways that produce metabolites involve a series of steps catalysed by a number of enzymes. Each enzyme could be targeted by a different ligand to produce a greater effect on the flux through the pathway than would be produced by any one. A further benefit of using combinations of agents is that it would prevent the development of resistance to the therapy: it is a major problem in the use of antibiotics and anticancer agents that, after a time, resistance develops as a result of mutation. If the agents comprised a mixture that targeted different molecules in the cell, multiple mutations would be required to produce resistance. Clearly, the chance of a cell undergoing two random mutations that coincide two produce resistance to two agents is much less likely than the single mutation required to overcome the effects of a single agent.
THE INVENTION
The invention provides a method of comparing ligands which method comprises: providing a target which is a polymeric molecule having an intramolecular structure, and a plurality of different ligands in the form of an array on a solid surface; applying the target in solution to the array of ligands; and observing quantitative differences between interactions of the target with different ligands of the array; provided that, when the ligands are not oligonucleotides or oligonucleotide analogues, then the target is a nucleic acid.
The invention also provides a method of determining combinations of ligands specific for a target, which method comprises the steps of: solid surface, under conditions which allow interaction between the other ligands and the target complex, and complex.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is an illustration of an array of all tetranucleotides.
FIGS. 2(a) and (b) depict scanning arrays of oligonucleotide precursors applied in a circular patch.
FIG. 3 shows hybridisation of tRNA.sup.phe to an array of type N.sub.3 X.sub.2 N.sub.3.
FIG. 4 shows the effect of including non-radioactive oligonucleotides with the tRNA target in a hybridisation solution.
FIG. 5 shows the sites of hybridisation of cooperative antisense oligonucleotides to the structure of rabbit .beta.-globin mRNA.
FIG. 6 shows four arrays of complements to a region of human CFTR gene.
FIG. 7(a) shows a folded structure of the Rev response element (RRE) of HIV RNA derived from computer molecular modelling. FIGS. 7(b) and (c) show the hybridisation of labelled RRE HIV RNA to two universal arrays.
FIGS. 8(a), (b) and (c) show scanning arrays of the region of RRE analyzed in FIG. 7.
FIGS. 9(a) and (b) show the analysis described in FIG. 8 conducted to presence of neomycin.
FIG
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Southern et al., "Analyzing and Comparing Nucleic Acid Sequences by Hybridization to Arrays of Oligonucleotides: Evaluation Using Experimental Models" Genomics 13:1008-1017, 1992.
Case-Green Stephen Charles
Mir Kalim Ullah
Southern Edwin Mellor
Oxford Gene Technology Limited
Sisson Bradley L.
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