KN-PASA Aptamer Selection via Nuclease Digestion
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Solution Overview
Problem
Current methods for selecting aptamers, such as SELEX, are laborious and require sophisticated instrumentation, making them impractical for routine use in average laboratories, and struggle with efficiently identifying aptamers with low binding affinity due to the mass action problem of weak binders competing with strong binders.
Innovation Solution
The method of Knockdown Nuclease Protected Aptamer Selection and Acquisition (KN-PASA) uses nucleases to separate target-bound aptamers from unbound oligonucleotides without physical separation, leveraging the resistance of bound aptamers to hydrolysis for enrichment, allowing for aptamer selection in one to three cycles using standard lab equipment like a PCR thermocycler and centrifuge.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If SELEX method is used for aptamer selection, then aptamers can be identified with high binding affinity, but the process becomes laborious and requires sophisticated instrumentation
Solution Approach 1:
The patent replaces the mechanical/physical separation system of SELEX with an enzymatic system. Instead of using complex instrumentation for physical separation of bound and unbound oligonucleotides, the invention uses nucleases to selectively digest unbound oligonucleotides, leaving bound aptamers intact. This enzymatic approach simplifies the device requirements while maintaining selection precision.
Solution Approach 2:
The patent introduces nucleases as intermediary agents to facilitate the separation process. These enzymes act as mediators that selectively recognize and digest unbound oligonucleotides based on their lack of protective binding, thereby enabling aptamer enrichment without complex separation instrumentation.
2Manufacturing precision
If SELEX method is used for aptamer selection, then specific aptamers can be obtained, but the process requires multiple laborious cycles
Solution Approach 1:
The replacement of mechanical separation with enzymatic digestion significantly accelerates each selection cycle. The nuclease-based approach rapidly eliminates unbound oligonucleotides in a single treatment step, eliminating the time-consuming repeated physical separation cycles required by SELEX while maintaining selection specificity.
3Measurement precision
If traditional selection methods are used, then strong binders can be identified, but weak binders are lost due to mass action competition
Solution Approach 1:
The patent extracts or removes the competitive mass action effect that plagues traditional SELEX. By using nucleases to selectively digest unbound oligonucleotides regardless of the binding strength of other molecules in the pool, the method eliminates the competitive suppression of weak binders. This allows weak aptamers to be enriched and identified that would otherwise be lost in the mass action competition of conventional methods.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
KN-PASA enables efficient and rapid identification of aptamers with high binding affinity and specificity, overcoming the limitations of existing methods by reducing the need for specialized equipment and allowing for the selection of weaker binders, thus improving the aptamer selection process.
Implementation Method 1
digesting unbound nucleic acid molecules with one or more endonucleases, with one or more exonucleases, or with one or more endonuclease in combination with one or more exonucleases
Data Source
AI summary
A method for identifying aptamers that bind to target molecules may include contacting an oligonucleotide library with target molecule and digesting unbound oligonucleotides with one or more endonucleases, one or more exonucleases, or one or more endonucleases in combination with one or more exonucleases. A method for identifying aptamers may further include optionally subjecting selected aptamers to one or more rounds of selection under conditions of increased stringency. A method for identifying aptamers may include yet further amplifying selected aptamers. The described methods may be performed in a screen for identifying aptamers either alone or in combination with other methods typically employed in the art for selecting aptamers (such as, e.g., SELEX). Also contemplated herein are systems and kits for accomplishing the above.