3' Aptamer Modification for Nuclease-Resistant NGS Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing aptamer-based assays face challenges in accurately quantifying protein expression due to variations in protein concentrations within and between biological samples, and there is a need for improved methods to generate sequencing libraries from aptamers for next-generation sequencing (NGS) that overcome nuclease degradation.
Innovation Solution
Aptamer detection methods involving exonuclease-mediated deprotection of the 3' end, followed by modification and capture, or hybridization with oligonucleotides to generate sequencing libraries, using reporter probes with unique identification sequences for aptamer identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If aptamers are used for high-affinity binding to analytes, then detection specificity is improved, but vulnerability to nuclease degradation increases
Solution Approach 1:
The patent employs composite nucleic acid structures combining aptamer sequences with protective elements. Specifically, aptamers are constructed with modified nucleotides (e.g., phosphorothioate linkages, 2'-O-methyl modifications) that create a composite structure resistant to nuclease degradation while preserving the aptamer's binding function. This composite approach allows the molecule to simultaneously achieve high analyte affinity and nuclease resistance.
Solution Approach 2:
The patent implements protective measures before nuclease degradation can occur by incorporating stable nucleic acid frameworks and protective coatings during aptamer synthesis. The aptamers are designed with inherent structural stability and protective chemical modifications that cushion them against enzymatic degradation throughout the assay process, ensuring reliability from sample collection through detection.
2Adaptability or versatility
If multiplexed aptamer assays are implemented to characterize multiple analytes, then assay versatility is improved, but difficulty in identifying useful detection ranges increases
Solution Approach 1:
The patent employs universal detection mechanisms that work across multiple analytes simultaneously. Reporter probes with standardized detection domains and uniform signal transduction pathways enable consistent measurement across diverse analytes. This universal approach allows the same detection system to handle multiple analytes with different concentration ranges, simplifying the identification of useful detection ranges for each analyte within the multiplexed assay.
Solution Approach 2:
The patent incorporates internal reference standards and control elements that provide feedback on detection performance for each analyte. By including known concentrations of reference analytes and monitoring signal responses, the system automatically identifies useful detection ranges for each target analyte in the multiplexed assay, even when concentration variations are high.
3Measurement precision
If aptamer sequences are optimized for binding affinity, then binding specificity is improved, but challenges for downstream detection workflows increase
Solution Approach 1:
The patent divides the aptamer molecule into functional segments: a binding domain optimized for high analyte affinity and a detection domain with standardized sequences for downstream processing. This segmentation allows the binding-optimized region to maintain high specificity while the standardized detection region facilitates easy integration with downstream workflows such as PCR amplification, sequencing, or fluorescence detection.
Solution Approach 2:
The patent introduces intermediary reporter probes that bridge the aptamer-analyte complex and the detection system. These reporter probes hybridize to the aptamer and provide standardized detection interfaces, mediating between the binding-optimized aptamer sequence and the downstream detection workflow. This intermediary approach preserves binding specificity while simplifying downstream operations.
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
Enables accurate quantification of aptamers and their targets through NGS, overcoming nuclease resistance and facilitating efficient library preparation for aptamer-based assays.
Implementation Method 1
contacting the individual aptamer with an exonuclease to deprotect a 3′ end of the individual aptamer to generate a deprotected 3′ end of the individual aptamer
Implementation Method 2
hybridizing an oligonucleotide to the individual aptamer, wherein the oligonucleotide comprises a nonhybridizing 5′ region
Implementation Method 3
extending an oligonucleotide 3′ end to generate an extended strand
Data Source
AI summary
Aptamer detection techniques are described that may include aptamer modification to facilitate incorporation of adapter sequences. In an embodiment, a 3′ end of an aptamer may be modified by deprotection and subsequent ligation to the deprotected 3′ end or extension of the deprotected 3′ end. The modifications at the 3′ end of the adaptor may include adaptor sequences used for library preparation of a sequencing library.