Synthetic ssRNA Binding Assay for Therapeutic Nucleic Acid Detection
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Solution Overview
Problem
Determining the effective dose of therapeutic nucleic acids in vivo is challenging due to variability in uptake by target cells and tissues, making it difficult to assess their presence and functionality in biological samples.
Innovation Solution
The use of synthetic single-stranded ribonucleic acid (ssRNA) that is complementary to therapeutic nucleic acids, which binds and is subsequently cleaved by enzymes like RNase H or Argonaute protein, allowing for detection and quantification through amplification reactions, indicating the presence and functionality of the therapeutic nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional detection methods are used to determine therapeutic nucleic acid presence, then the detection process becomes complex and unreliable, but the patent introduces a synthetic ssRNA binding assay that simplifies and robustifies the detection
Solution Approach 1:
The patent introduces synthetic single-stranded RNA (ssRNA) as an intermediary probe that specifically binds to therapeutic nucleic acids of interest. This intermediary molecule mediates the detection process by forming a detectable complex with the target therapeutic nucleic acid, enabling reliable detection through binding assays without requiring complex conventional detection methodologies
Solution Approach 2:
The patent replaces complex conventional detection mechanisms with a simpler biochemical binding assay. Instead of using elaborate detection systems, the method relies on the fundamental biochemical principle of complementary base pairing between synthetic ssRNA and therapeutic nucleic acids, followed by detection of the binding event through enzymatic cleavage or other straightforward assays
2Measurement precision
If therapeutic nucleic acids are delivered in vivo to target cells, then the therapeutic effect is achieved, but it becomes difficult to determine uptake and effective dosage
Solution Approach 1:
The synthetic ssRNA acts as a detectable intermediary that binds specifically to the therapeutic nucleic acid. By detecting the presence and amount of bound ssRNA-therapeutic nucleic acid complexes, the method enables precise measurement of therapeutic nucleic acid uptake and dosage in biological samples
Solution Approach 2:
The detection method utilizes detectable changes resulting from the binding event, such as enzymatic cleavage of the ssRNA or other signal-generating changes that indicate the presence and quantity of therapeutic nucleic acids, enabling precise quantification
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
This method provides a robust and quantitative means to detect and quantify therapeutic nucleic acids, such as DNA and RNAi agents, by evaluating their ability to bind with synthetic ssRNA, thereby determining their presence and functionality in biological samples.
Implementation Method 1
a synthetic single-stranded ribonucleic acid (ssRNA) that is longer than and contains a region that is complementary to the therapeutic nucleic acid
Implementation Method 2
an enzyme that binds to double-stranded nucleic acid and is capable of mediating cleavage of the synthetic ssRNA
Implementation Method 3
performing at least one assay to detect cleavage of the synthetic ssRNA, wherein the assay to detect cleavage of the synthetic ssRNA comprises amplifying a reverse transcript of the synthetic ssRNA
Data Source
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AI summary
The present disclosure relates, in some aspects, to the field of nucleic acid detection. Disclosed herein are methods and compositions for detecting nucleic acids using synthetic single-stranded ribonucleic acids (RNAs). In certain embodiments, synthetic single-stranded RNAs are used to detect therapeutic nucleic acids, such as therapeutic deoxyribonucleic acids (DNAs) and/or therapeutic ribonucleic acids (RNAs).