RNA Aptamer Gradient Elution Screening for Low False Positives
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Solution Overview
Problem
Existing RNA aptamer screening techniques suffer from high false positive rates, non-optimal binding ability, high time cost, poor reproducibility, and require manual operation, hindering their efficient application in RNA drug development.
Innovation Solution
A method involving a library of RNA aptamers incubated with a solid carrier, using buffer gradients for elution, reverse-transcribing and high-throughput sequencing to identify high-affinity sequences, and analyzing binding potential to optimize screening conditions and reduce false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional RNA aptamer screening techniques are used, then screening can be performed, but the false positive rate is high and binding ability is non-optimal
Solution Approach 1:
The screening process is divided into multiple elution steps with different buffer conditions. Each elution step separates aptamers based on their binding strength to the target, allowing systematic evaluation of binding potential and reduction of false positives through multi-stage filtering
Solution Approach 2:
The method employs gradient elution with progressively changing buffer conditions (increasing salt concentration, changing pH, or adding competing ligands) to systematically elute aptamers based on their binding affinity, enabling precise ranking and selection of high-affinity sequences
2Productivity
If traditional screening methods are used, then aptamers can be identified, but the time cost is high requiring 10-16 rounds of repetitive screening
Solution Approach 1:
The method performs continuous elution and sequencing in a single experimental run, eliminating the need for multiple discrete screening rounds. The gradient elution continuously separates and enriches aptamers while sequencing continuously identifies high-affinity sequences, dramatically reducing the 10-16 rounds required by traditional methods
Solution Approach 2:
The method replaces mechanical repetitive manual screening with automated high-throughput sequencing and bioinformatic analysis. Once the RNA aptamer library is bound to the target, automated sequencing and computational evaluation replace the manual iterative screening process, reducing time from months to days
3Reliability
If manual operation is required, then screening can be performed, but reproducibility is poor and operations are complex
Solution Approach 1:
The method establishes a universal gradient elution protocol that can be applied to various target molecules and aptamer libraries using the same basic workflow. The standardized multi-step elution and sequencing protocol enables reproducibility across different laboratories and reduces dependency on manual optimization
Solution Approach 2:
The method employs automated sequencing and bioinformatic tools that self-evaluate and rank aptamers based on binding potential without requiring manual interpretation. The automated analysis system consistently applies the same criteria across all samples, ensuring reproducibility and reducing human error in the evaluation process
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
The method reduces false positive rates, enhances screening ability, shortens experiment time, improves reproducibility, and simplifies operations, enabling rapid identification of high-affinity RNA aptamers for applications like live cell imaging and SARS-COV-2 polymerase inhibition.
Implementation Method 1
incubating the library of step 1) with a solid carrier fixed with a target, thereby inducing the RNA aptamer in the library to sufficiently bind to the target
Implementation Method 2
adopting a buffer gradient to elute the RNA aptamers bound to the target on the solid carrier in step 2), and collecting the eluate for each elution, respectively
Implementation Method 3
reverse-transcribing the RNA aptamers obtained in step 5) to obtain cDNAs
Data Source
AI summary
The present invention provides a method for screening an aptamer from an RNA library. According to the method, an eluent eluted each time is collected and a specific elution program is combined at the same time, thereby not losing any information of the RNA aptamer and achieving the technical effects of low false positive rate, high binding capacity of the screened aptamer, short library preparation time, capability of performing only one round of enrichment, high library preparation repeatability and suitability for an automatic mechanical arm.