Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Assignees
Biotex Inc • Base Pair Biotechnologies Inc
Member
Base Pair BiotechnologiesBase Pair Biotechnologies specializes in custom aptamer discovery and development for research, diagnostics, therapeutics, and industrial applications. The company leverages proprietary multiplex selection, advanced bioinformatics, and chemical modification techniques to develop high-affinity and selective nucleic acid aptamers. Base Pair enables affinity reagent development, biosensor design, and molecular detection for a broad range of targets and partners across academia and industry.
Base Pair Biotechnologies specializes in custom aptamer discovery and development for research, diagnostics, therapeutics, and industrial applications. The company leverages proprietary multiplex selection, advanced bioinformatics, and chemical modification techniques to develop high-affinity and selective nucleic acid aptamers. Base Pair enables affinity reagent development, biosensor design, and molecular detection for a broad range of targets and partners across academia and industry.
Publication Number
US-8314052-B2
Publication Date
2012-11-20
Expiration Date
Abstract
The present invention relates to methods for generating functional biomolecules. In one exemplary aspect of the invention, generation of functional biomolecules may be performed against multiple targets simultaneously within a single system. In general, a plurality of targets may be disposed within in a single reaction volume and a library of biomolecules, such as a nucleic acid library, may be applied to the reaction volume. The members of the library that do not bind to any of the plurality of targets under given conditions may then be partitioned. The remaining members of the library may then be marked and/or tagged, such as to identify the particular target or targets to which the member of the library binds. The binding members of the library may then be isolated and, by virtue of the marking or tagging, be matched to a particular target or targets.
Core Innovation
The present invention relates to methods for generating functional biomolecules, particularly to methods for generating numerous different functional nucleic acids against multiple target molecules simultaneously. In general, a plurality of targets may be disposed within a single reaction volume and a library of biomolecules, such as a nucleic acid library, may be applied to the reaction volume. Members of the library that do not bind to any of the plurality of targets under given conditions may be partitioned, and remaining members that bind to the plurality of targets may then be marked and/or tagged to identify the particular target or targets to which the member binds.
The invention addresses the conventional approach in which SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target after multiple rounds of selection. SELEX typically requires multiple rounds to identify aptamers for individual targets, and the present methods provide simultaneous generation of functional biomolecules against multiple targets within a single system to increase throughput and multiplex capacity.
In an exemplary embodiment, a plurality of target molecules are affixed to a substrate within a single reaction volume as an array with ordered deposition of targets in clusters or spots, a library of nucleic acids is contacted with the array, non-binding members are partitioned, and the binding members are marked or tagged with a plurality of identifiers such that the nucleic acids bound to a particular target spot may be identified by the sequence of an associated identifier. Identifiers may include unique or semi-unique sequences which correspond to spatial locations on the array and may be incorporated into amplified products to associate a given library member with a particular target while preserving the sequence of the library member.
Claims Coverage
One independent claim was identified with seven main inventive features.
Simultaneous selection on an array
Contacting a library containing aptamers comprising a diversity of nucleic acids each having a randomized region and a constant region, with a plurality of different targets simultaneously in a single reaction volume, each of said targets being fixed at a different predetermined location on an array.
Partitioning non-binding library members
Partitioning members of said library that do not bind to said plurality of different targets.
Marking binding members with target-indicative identifiers
Marking the members of the library that bind to the plurality of different targets with identifiers, each of said identifiers being indicative of the particular target.
Applying a different identifier to each location
Applying a different identifier to each of said different predetermined locations on said array.
Identifier composition with hybridizing and primer regions
Identifiers comprising a single-stranded DNA oligonucleotide having a variable region which identifies one of said predetermined locations on said array, said variable region being flanked by a first constant region having a sequence for hybridizing to said constant region of said library, and a second constant region having a primer binding site.
Identifying and correlating binding members via identifiers
Identifying the binding members of the library and correlating them to the targets via said identifiers.
Amplification to correlate aptamer sequence to target
Performing a nucleic acid amplification reaction on each of said hybridized identifiers and said aptamers to generate an amplification product wherein said amplification product correlates the sequence of one of said aptamers to one of said targets to which said aptamer binds.
The independent claim covers simultaneous, multiplexed selection of aptamers on an array by partitioning non-binders, marking binders with location-indicative single-stranded DNA identifiers that hybridize to library constant regions and contain primer sites, identifying binding members via those identifiers, and producing amplification products that correlate aptamer sequences to their targets.
Stated Advantages
High capacity, multiplexed identification procedures may save time, expense, and physical space for the process over single target identification processes.
May be utilized to identify and/or eliminate biomolecules that bind or have a tendency to bind to multiple targets.
Associating a given member with a target while preserving the particular sequence of the member by appending a locational identifying sequence, which is useful for resolving multiple binders to a single target or members that bind multiple targets.
Documented Applications
Generating functional nucleic acids (aptamers) against multiple target molecules simultaneously in a single reaction volume or on target arrays.
Use of aptamers as sensors, therapeutic tools, to regulate cellular processes, and to guide drugs to their specific cellular target(s).
Application to target arrays and histology sections as target sets for producing affinity binding nucleic acids corresponding to specific locations or features.
Generating and tagging peptide ligands via display methods (including phage display, mRNA display, ribosome display, yeast display, bacterial display) with associated nucleic acid sequences for target identification.
Handling, sorting and sequencing resultant sequences from multiplexed binding processes, including use of parallel sequencing methods to identify aptamers and using sequence sorting/alignment to identify shared features.
Monitoring and controlling library diversity during selection using Cot analysis to measure sequence diversity.
Interested in licensing this patent?