In Situ Circular Oligonucleotide Amplification Around RNA Secondary Structures
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Solution Overview
Problem
Existing methods struggle to efficiently form and amplify circular oligonucleotides due to secondary structures in target sequences, such as hairpin loops and pseudoknots, which hinder polymerase progression and ligation, limiting detection and amplification of target nucleic acid sequences.
Innovation Solution
A method involving a blocking oligonucleotide and a probe oligonucleotide are used to form a circular oligonucleotide by hybridizing to flanking sequences of a target polynucleotide, extending with a polymerase, and ligating the extension strand to the probe oligonucleotide, followed by amplification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional amplification methods are used on target sequences with secondary structures, then amplification efficiency is reduced, but the complexity of the method increases when attempting to overcome these structures
Solution Approach 1:
The method divides the target sequence into two separate binding regions for the probe oligonucleotide - one region binds to the first flanking sequence and another region binds to the second flanking sequence. This segmentation allows the probe to effectively capture and amplify the target sequence even in the presence of secondary structures, improving amplification efficiency without requiring complex additional steps
Solution Approach 2:
The probe oligonucleotide serves as an intermediary molecule that bridges the two flanking sequences of the target. By hybridizing to both flanking sequences simultaneously, the probe mediates the formation of a circular structure that enables efficient amplification, simplifying the overall process while overcoming secondary structure barriers
2Speed
If polymerase extension is attempted through secondary structures like hairpin loops and pseudoknots, then polymerase progression is hindered, but increasing enzyme strength or temperature may denature the target
Solution Approach 1:
The probe oligonucleotide performs preliminary binding to both flanking sequences before polymerase extension begins. This preliminary action creates a stable circular structure that guides the polymerase along the correct path, preventing it from becoming stalled by secondary structures like hairpin loops and pseudoknots, thus maintaining both progression speed and target integrity
Solution Approach 2:
The method replaces reliance on polymerase mechanical strength to force through secondary structures with a structural guidance approach. The circular probe structure provides a predetermined path that substitutes for brute-force polymerase action, allowing smooth progression without denaturation while maintaining target integrity
3Productivity
If ligation is performed in the presence of secondary structures, then ligation efficiency decreases, but extending reaction time increases the risk of non-specific products
Solution Approach 1:
The method changes the structural parameters of the substrate by forming a circular probe-oligonucleotide structure that encompasses the target sequence. This parameter change creates an optimal configuration for ligation, where the 5' and 3' ends are properly positioned and oriented, dramatically improving ligation efficiency while maintaining short reaction times and preventing non-specific products
4Measurement precision
If circular oligonucleotides are formed and amplified, then detection sensitivity increases, but the complexity of forming the circular structure initially increases
Solution Approach 1:
The method merges the binding functions of two separate probes into a single probe oligonucleotide that simultaneously binds to both flanking sequences. This merging simplifies the formation process of the circular structure while maintaining the detection sensitivity benefits, as the single probe efficiently captures and circularizes the target in one step rather than requiring multiple sequential 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
This approach enables effective formation and amplification of circular oligonucleotides, allowing for robust detection and sequencing of target nucleic acid sequences despite secondary structures, enhancing the resolution of overlapping RNA transcripts.
Implementation Method 1
contacting a target polynucleotide complex with a probe oligonucleotide, thereby forming a probe polynucleotide complex
Implementation Method 2
extending the probe oligonucleotide along the target sequence with a polymerase to generate an extension strand including a complementary sequence of the target sequence
Implementation Method 3
ligating the extension strand to the probe oligonucleotide hybridization sequence, thereby generating a circular oligonucleotide
Implementation Method 4
amplifying the circular oligonucleotide by extending an amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase, thereby generating an amplification product including multiple copies of the target sequence
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods of use thereof for amplifying polynucleotides within cells and tissues.