Poly A Polymerase Reactivity with Modified Oligonucleotides
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Solution Overview
Problem
The existing PALSAR method struggles to detect oligonucleotides or DNA with chemically modified nucleic acid bases at the 3′-end due to low signal and high background values when using Mg2+ concentrations, particularly in biological samples like blood plasma or brain, resulting in low detection sensitivity.
Innovation Solution
Adjusting the Mn2+ concentration during the poly A polymerase reaction enhances the reactivity and sensitivity of the detection method by increasing the signal-to-noise ratio, allowing for effective detection of oligonucleotides with chemically modified 3′-end nucleic acid bases, even in challenging biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Mg2+ concentration is used for poly A polymerase reaction, then the reaction can proceed, but the detection sensitivity is low due to low signal and high background values
Solution Approach 1:
The patent changes the metal ion parameter from Mg2+ to Mn2+ in the poly A polymerase reaction. This parameter change fundamentally alters the enzyme's catalytic properties, enabling it to effectively process oligonucleotides with chemically modified 3'-end bases. The Mn2+ ion facilitates the polymerase reaction while producing a detectable signal with suppressed background, thereby resolving the contradiction between reaction reliability and measurement precision.
2Reliability
If poly A polymerase is used to add poly A to oligonucleotides with chemically modified 3'-end bases, then the oligonucleotides can be detected, but the reactivity is insufficient under conventional conditions
Solution Approach 1:
The patent applies parameter change by substituting Mn2+ for Mg2+ to enhance poly A polymerase reactivity toward chemically modified oligonucleotides. This parameter modification enables the enzyme to overcome the steric and electronic barriers presented by 3'-end modifications, achieving sufficient reaction productivity for reliable detection.
Solution Approach 2:
Mn2+ acts as an intermediary that mediates between the poly A polymerase and the chemically modified oligonucleotide substrate. The metal ion facilitates the formation of the enzyme-substrate complex and stabilizes the transition state, enabling the polymerase to process modified bases that would otherwise be refractory to enzymatic action.
3Ease of operation
If conventional poly A polymerase reaction conditions are used, then the reaction is simple, but the background noise is high making detection difficult
Solution Approach 1:
The patent maintains operational simplicity by using a single metal ion substitution (Mg2+ to Mn2+) rather than complexing the reaction protocol. This parameter change inherently suppresses background noise while preserving the ease of operation, as the Mn2+-dependent polymerase reaction proceeds under similarly simple conditions to conventional Mg2+-based reactions.
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 significantly improves the detection sensitivity of oligonucleotides with chemically modified 3′-end nucleic acid bases, enabling reliable detection in biological samples by suppressing background noise and enhancing signal strength.
Implementation Method 1
bringing poly A polymerase into contact with the target oligonucleotide in the sample in the presence of Mn2+
Implementation Method 2
it was found that by adjusting the Mn2+ concentration during a poly A polymerase reaction, it is possible to increase the signal and suppress the background
Implementation Method 3
the probes are labelled, and these form a mesh-like large DNA lump by repeating a self-aggregating reaction through hybridization
Implementation Method 4
a step of causing a capture probe to react with the microRNA to which the poly A is added to capture the microRNA
Data Source
AI summary
An object of the present invention is to increase the reactivity of poly A polymerase with an oligonucleotide or a DNA in which the nucleic acid base at the 3′-end is chemically modified, and then to improve the sensitivity of detection through a PALSAR method or the like. The present invention relates to increasing the reactivity of poly A polymerase with an oligonucleotide or a DNA used for the nucleic acid medicine, in which the nucleic acid base at the 3′-end is chemically modified, and then adjusting the Mn2+ concentration during a poly A polymerase reaction, in a method of detecting the oligonucleotide, for a detection target that is an oligonucleotide or a DNA in which the nucleic acid base at the 3′-end is chemically modified, through steps of addition of poly A to the 3′-end of the oligonucleotide with poly A polymerase, capturing with a capture probe, and amplification by a PALSAR method or the like. Thus, the problem of the present invention is solved.