Thermostable Polymerase Aptamers for Reversible Hot-Start Control
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Solution Overview
Problem
Existing nucleic acid synthesis assays using thermostable polymerases face issues with mispriming and non-specific product generation due to primer hybridization at lower temperatures, which can deplete reaction mixtures and mask the target product.
Innovation Solution
Development of aptamers that form a secondary structure to reversibly inhibit thermostable polymerases at lower temperatures, dissociating at optimal reaction temperatures to allow primer extension and target product generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the assay is performed at lower or ambient temperature, then primer hybridization occurs more readily, but mispriming and non-specific product generation increase
Solution Approach 1:
The patent applies preliminary anti-action by using an aptamer that pre-binds to the thermostable polymerase at ambient temperatures, preventing the polymerase from extending misprimed primers. The aptamer-polymerase complex is formed before the PCR reaction begins, and this complex actively prevents harmful mispriming events during the annealing step. Upon heating to the extension temperature, the aptamer releases the polymerase, allowing specific product amplification to proceed.
2Object-generated harmful factors
If chemical inhibitors are used to prevent mispriming, then non-specific product generation is reduced, but the reaction mixture composition becomes more complex
Solution Approach 1:
The patent employs an aptamer as an intermediary molecule that mediates between the polymerase and the primers. The aptamer serves as a reversible inhibitor that can be easily added to the reaction mixture without significantly altering its overall composition. The aptamer acts as a controllable switch: bound to polymerase at low temperatures to prevent mispriming, and released at high temperatures to allow specific amplification, thereby reducing non-specific products without complicating the reaction mixture.
3Productivity
If the polymerase is actively present at lower temperatures, then primer extension can occur, but non-specific amplification is generated
Solution Approach 1:
The patent applies dynamics by making the polymerase activity dynamically controllable through temperature-dependent aptamer binding. At ambient temperatures, the aptamer is bound to the polymerase, dynamically suppressing its activity and preventing non-specific amplification. When the temperature is raised to the extension temperature, the aptamer dynamically releases the polymerase, enabling efficient and specific primer extension. This dynamic control allows the same polymerase to serve both protective and productive functions at different stages of the PCR cycle.
Data Source
AI summary
The present disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.