Single-Cycle Aptamer Selection Using DNase I Digestion
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Solution Overview
Problem
Current methods for selecting aptamers with high affinity and specificity for proteins, such as SELEX, often require multiple cycles and are limited by sample volume and sequence diversity, making them inefficient and not suitable for common laboratory practices.
Innovation Solution
A DNase I mediated single-cycle aptamer selection method that uses membrane partitioning and western-blotted proteins to isolate strong binding aptamers from crude protein extracts, allowing for the use of small target protein amounts and integrating with gel electrophoresis separation techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple cycles of SELEX are used to select aptamers with high affinity and specificity, then the selection stringency and aptamer quality are improved, but the time and resources required increase significantly
Solution Approach 1:
The patent segments the SELEX process into distinct functional steps: (1) incubation of DNA library with target protein on membrane, (2) washing to remove unbound sequences, (3) DNase I digestion to degrade unbound/weakly bound sequences, and (4) elution and PCR amplification of bound sequences. This segmentation allows each step to be optimized independently, achieving high selection stringency in a single cycle rather than requiring multiple repetitive cycles.
Solution Approach 2:
The patent introduces DNase I as an intermediary enzyme that selectively degrades unbound and weakly bound DNA sequences while leaving protein-bound sequences intact. This intermediary mechanism enables efficient removal of non-specific binders in a single step, dramatically improving selection stringency without requiring multiple cycles of partitioning and amplification.
2Productivity
If capillary electrophoresis SELEX is used to separate protein-bound sequences from unbound sequences, then single-cycle selection is achieved, but over 97% of DNA sequence diversity is lost
Solution Approach 1:
The patent uses PCR amplification as a copying mechanism to replicate and enrich the bound DNA sequences after selection. By amplifying the sequences that remained bound to the protein during the single selection cycle, the method preserves and even enhances sequence diversity while achieving rapid selection. This copying step compensates for any sequences that may have been lost during the separation process.
Solution Approach 2:
The patent changes the selection parameter from physical separation (capillary electrophoresis) to enzymatic degradation (DNase I digestion). This parameter change allows for selective removal of unbound sequences while preserving bound sequences and their diversity. The method also utilizes membrane binding properties and washing conditions as selectable parameters to enrich for high-affinity binders.
3Reliability
If magnetic microfluidic chip SELEX is used to expose small quantity of target proteins to DNA library, then extremely high selection stringency is achieved, but the method is limited to microscale sample volumes and requires immobilization of target molecules
Solution Approach 1:
The patent creates a universal selection platform that can accommodate various sample volumes and protein types by using a membrane-based system. The membrane can be incubated with different amounts of protein (nanogram to microgram range) and the same DNase I-based selection protocol can be applied. This multi-functional approach eliminates the need for protein immobilization on magnetic beads and allows flexibility in sample volume.
Solution Approach 2:
The patent uses the membrane as an intermediary carrier that presents target proteins to the DNA library in a controlled manner. The membrane surface acts as a platform for protein binding, allowing selective interaction without requiring magnetic beads or microfluidic channels. This intermediary approach enables scalability from microscale to macroscale sample volumes while maintaining selection stringency.
4Reliability
If conventional SELEX is used to select aptamers, then high affinity and specificity can be achieved, but the process requires 8-20 rounds of partitioning, separation, and PCR amplification
Solution Approach 1:
The patent merges multiple SELEX steps into a single integrated cycle: (1) binding incubation, (2) washing, (3) DNase I digestion, and (4) elution/PCR are combined into one selection round. This merging eliminates the need for 8-20 separate partitioning and amplification cycles while maintaining the ability to select high-affinity and specific aptamers through the synergistic action of these combined steps.
Solution Approach 2:
The patent introduces DNase I as a key intermediary that enables the consolidation of multiple selection steps into one cycle. The enzyme provides a rapid and specific means of removing unbound sequences, replacing the need for repeated physical separation and amplification cycles. This intermediary mechanism dramatically reduces the number of process steps while maintaining selection effectiveness.
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
This method enables the rapid selection of thermally stable and modifiable aptamers with high affinity for proteins, such as the HBV core protein, in a single cycle, preserving sequence diversity and being applicable to multiple proteins in cell lysates or mixtures, with potential for clinical and diagnostic applications.
Implementation Method 1
digesting the unbound and weakly bound nucleic acids with a nuclease
Implementation Method 2
proteins blotted on a membrane
Implementation Method 3
integrating with gel electrophoresis separation techniques
Data Source
AI summary
A method for single-cycle selection of aptamers is provided. More specifically, a method comprising single-cycle selection of aptamers for proteins blotted on a membrane is provided. In some embodiments, the present methods can comprise a deoxyribonuclease I (DNase I) mediated aptamers selection strategy that may be capable of isolating strong binding aptamers for target proteins from a crude protein extract. Aptamers selected using the present method are further provided. More specifically, the presently selected aptamers may be thermally stable, modifiable and easily produced through single-cycle synthesis process. The present aptamers may provide unique affinity reagents for use in diagnosing and detecting infectious disease (for example Hepatitis B), for research and biochemical studies (e.g. into molecular mechanisms). Further, the present aptamers may be utilized to develop unique assays or kits for clinical application, such as monitoring disease treatment and outcome.