Circularizable Probes for In Situ Nucleic Acid Analysis
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Solution Overview
Problem
Current methods for analyzing nucleic acids in biological samples face challenges such as low assay specificity, high rates of false positive results, and limitations in the length of oligonucleotide synthesis, which restricts the production of probes with multiple barcode sequences.
Innovation Solution
The method involves contacting a biological sample with a templated ligation probe that hybridizes to a target nucleic acid, followed by ligation to form a ligated probe. This probe is then circularized using a selector probe and undergoes rolling circle amplification to generate a detectable product.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If templated ligation is used to improve assay specificity, then false positive results are reduced, but the complexity of the procedure increases due to additional ligation steps
Solution Approach 1:
The probe is divided into multiple segments (first part, second part, and optional third part) that are sequentially ligated to the target nucleic acid. This segmentation allows the templated ligation to occur in controlled steps, improving assay specificity by requiring multiple matching events while managing procedural complexity through systematic progression of ligation steps.
2Loss of information
If oligonucleotide synthesis length is increased to accommodate more barcode sequences, then probe information capacity increases, but synthesis feasibility and accuracy decrease
Solution Approach 1:
The barcode capacity is expanded by using multiple separate oligonucleotide parts (first part, second part, third part) rather than synthesizing one extremely long oligonucleotide. Each part can be synthesized within standard length limits, then ligated together to form a complete probe with high information capacity that would be impossible to synthesize as a single piece.
Solution Approach 2:
Multiple oligonucleotide parts are nested within a ligation framework where shorter, synthesizable segments are assembled into a longer functional probe structure. The first part, second part, and third part are sequentially joined through templated ligation, creating a nested assembly process that overcomes synthesis length limitations.
3Productivity
If circularization of the probe is performed to enable rolling circle amplification, then signal amplification is enhanced, but the risk of circularization of non-specific probes increases
Solution Approach 1:
The probe is first ligated to the target nucleic acid in a templated manner before circularization occurs. This preliminary ligation step ensures that only probes that have successfully bound to their complementary target sequences are circularized and subsequently amplified, preventing non-specific probes from generating false positive signals.
Solution Approach 2:
The templated ligation step acts as a feedback mechanism that verifies probe-target complementarity before allowing circularization. The ligation efficiency provides feedback on the correctness of probe binding, ensuring that only properly matched probes proceed to the amplification stage, thereby reducing false positives while maintaining high signal amplification.
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 enhances assay specificity, reduces false positive results, and allows for the production of probes with longer barcode sequences, thereby improving the analysis of nucleic acids in biological samples.
Implementation Method 1
the first part and the second part hybridize to the target nucleic acid such that ligatable ends of the first and second parts are juxtaposed for ligation
Implementation Method 2
ligating the first and second parts using the target nucleic acid as a template, thereby generating a ligated probe
Implementation Method 3
contacting the biological sample with a selector probe comprising a first strand and a second strand that form a partially double stranded probe
Implementation Method 4
circularizing the ligated probe using the selector probe as a template, thereby generating a circularized ligated probe
Implementation Method 5
performing rolling circle amplification of the circularized ligated probe in the biological sample to generate a rolling circle amplification product
Data Source
AI summary
The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.