Focused Aptamer Library Construction via Laser SELEX and DeSELEX
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Solution Overview
Problem
Current SELEX methods face challenges in efficiently constructing focused aptamer libraries against complex mixtures of native biomolecules, such as proteomes, which hinders the isolation of aptamers with desired biological activities due to sequence complexity and the need for multiple rounds of binding, partitioning, and amplification.
Innovation Solution
The method involves constructing focused libraries by contacting an initial collection of single-stranded nucleic acids with a mixture of native biomolecules, partitioning bound and unbound nucleic acids, amplifying the bound ones, and repeating the process to reduce library complexity, using techniques like Laser SELEX to enrich specific aptamers and DeSELEX to deplete non-specific ones, allowing for easier screening and identification of aptamers with desired biological activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a library with many nucleic acids of different sequences is constructed to achieve structurally diverse conformations, then the ability to select desired nucleic acids is improved, but the difficulty of selection increases due to low frequency of target-binding nucleic acids
Solution Approach 1:
The patent segments the selection process into multiple iterative rounds of binding, partitioning, and amplification. Each round focuses on enriching specific nucleic acid sequences that bind to the target, gradually reducing library complexity while maintaining diversity. This segmentation transforms an overwhelming single-step selection into manageable sequential steps.
Solution Approach 2:
The patent performs preliminary enrichment of target-binding nucleic acids through multiple rounds of selection before final identification. By pre-concentrating the desired sequences through iterative binding and partitioning, the method prepares the library in advance for easier detection and selection of high-affinity binders.
2Quantity of substance
If multiple rounds of binding, partitioning, and amplification are performed to enrich desired nucleic acids, then the frequency of target-binding nucleic acids is improved, but the time and complexity of the process increase
Solution Approach 1:
The patent implements continuous enrichment through iterative cycles where the output of one round (amplified nucleic acids) becomes the input for the next round of binding and partitioning. This continuous action progressively increases the frequency of target-binding nucleic acids without interruption, maximizing enrichment efficiency over time.
3Reliability
If frequent binders are enriched in the library, then the binding activity is improved, but the isolation of less abundant candidate aptamers becomes difficult due to masking
Solution Approach 1:
The patent applies partial enrichment by performing a limited number of selection rounds rather than exhaustive enrichment. This partial action allows moderate enrichment of high-affinity binders while preventing complete dominance that would mask lower-abundance aptamers, thus maintaining library diversity and enabling isolation of various candidate aptamers with different binding characteristics.
Data Source
AI summary
Focused aptamer libraries are constructed in accordance with a proteome (i.e., complex mixture of native biomolecules). The libraries may be screened to identify one or more candidate aptamers with desired biological activities other than specific binding to a target. Aptamers which are selected or derivatives thereof may be used for those specific activities in biological systems. Any combination of deconvoluting a focused library (functional profiling), increasing frequencies of particular aptamers in a focused library (Laser SELEX), and decreasing frequencies of particular aptamers in a focused library (DeSELEX) may be performed prior to assaying biological activity.