Oligonucleotide Quantification via Nuclease Digestion and PALSAR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods struggle to sensitively and specifically measure oligonucleotides, particularly distinguishing between intact and metabolized forms, especially at low concentrations, and are not suitable for multiplexing due to sequence-specific probe design requirements.
Innovation Solution
A method involving hybridization of target oligonucleotides with complementary nucleic acid probes, followed by single-strand-specific nuclease treatment to decompose incomplete hybridization products, allowing for the quantification of intact oligonucleotides using a PALSAR method or ELISA detection of labeled probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR method is used for quantification, then measurement can be performed, but measurement precision and quantitativity are insufficient
Solution Approach 1:
The patent replaces the semi-quantitative PCR method with a fully quantitative ELISA-based measurement system. This substitution transitions from a method requiring operator interpretation to one providing objective, quantifiable results through signal detection, thereby improving both measurement precision and operator independence.
Solution Approach 2:
The patent changes the detection parameter from qualitative/semi-quantitative PCR signal to quantitative ELISA signal measurement. This parameter change enables precise quantification of oligonucleotide concentrations while ensuring results are independent of operator skill and interpretation.
2Measurement precision
If conventional measurement system is used, then measurement can be performed, but inability to distinguish intact oligonucleotides from metabolites reduces measurement precision
Solution Approach 1:
The patent segments the measurement process into two distinct stages: first, ligation of intact oligonucleotides with detection probes, and second, S1 nuclease treatment to remove incomplete hybridization products and metabolites. This segmentation enables selective measurement of intact oligonucleotides while simplifying the overall measurement approach.
Solution Approach 2:
The patent extracts and removes metabolites and incomplete hybridization products from the measurement system through S1 nuclease treatment. This extraction step isolates the intact oligonucleotides for accurate quantification without requiring complex measurement systems.
3Measurement precision
If hybridization-ligation ELISA method is used, then specificity is improved, but device complexity increases and multiplexing becomes difficult
Solution Approach 1:
The patent creates a universal detection platform where a single probe design can detect multiple different oligonucleotide sequences. The standardized probe structure with universal flanking sequences enables multiplexing capability, allowing one measurement system to perform multiple detection functions simultaneously.
Solution Approach 2:
The patent changes the probe design parameter from sequence-specific throughout the entire probe to sequence-specific only in the central region, with universal flanking sequences. This parameter change maintains specificity for target detection while enabling multiplexing and reducing method complexity.
4Measurement precision
If measurement sensitivity is increased for low-concentration drugs, then detection capability is improved, but ability to maintain high quantitativity becomes difficult
Solution Approach 1:
The patent replaces PCR-based detection with ELISA-based detection, substituting a method that struggles with quantitativity at low concentrations with one that provides reliable quantitative results across a wide concentration range, thereby maintaining both sensitivity and quantitativity.
Solution Approach 2:
The patent changes the detection parameter and signal amplification mechanism from PCR to ELISA, enabling reliable quantification of low-concentration oligonucleotides while maintaining operator independence and high quantitativity through standardized signal measurement.
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 high sensitivity and specificity in measuring oligonucleotides, distinguishing between intact and metabolized forms, and supports multiplexing by improving the accuracy of oligonucleotide quantification in biological samples.
Implementation Method 1
hybridization of target oligonucleotides with complementary nucleic acid probes
Implementation Method 2
single-strand-specific nuclease treatment to decompose incomplete hybridization products
Data Source
AI summary
The present invention provides a method of detecting and a method of quantifying oligonucleotides with more excellent specificity and quantitativity as compared to conventional signal amplification (PALSAR) measurement methods.In the present invention, the problem is solved by hybridizing a target oligonucleotide to be measured with a complementary nucleic acid probe (3′-complementary sequence of target sequence-5′), or hybridizing the target oligonucleotide to be measured having given bases such as poly(A) added thereto with a complementary nucleic acid probe (3′-complementary sequence of target oligonucleotide+complementary sequence of given bases-5′), decomposing and removing an incomplete hybridization product by using a single-strand-specific nuclease such as Si nuclease, and measuring the nucleic acid probe contained in a remaining complete hybridization product by a PALSAR method.