Self-Complementary Stem Probe Sets for In Situ Nucleic Acid Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing oligonucleotide probe-based methods for in situ analysis of nucleic acids suffer from low sensitivity, specificity, and detection efficiency, requiring laborious optimization.
Innovation Solution
A method involving a probe set comprising a first and second polynucleotide that form a circularized probe through hybridization and ligation, using a self-complementary stem region to facilitate efficient rolling circle amplification and detection, without the need for a splint.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional oligonucleotide probe-based methods are used for in situ analysis, then the analysis can be performed, but the sensitivity, specificity, and detection efficiency remain low
Solution Approach 1:
The probe is divided into multiple modular regions (target binding region, stem region with self-complementary sequence, and ligation region). This segmentation allows each region to perform its specific function optimally, improving overall detection efficiency while maintaining manageable complexity through standardized modular design.
Solution Approach 2:
The probe structure incorporates nested functional elements where the stem region contains self-complementary sequences that fold back on themselves, and the overall probe structure is designed to be ligated into larger circular structures. This nesting improves detection efficiency by creating compact, stable structures that enhance signal generation.
2Productivity
If circularized probes are generated using traditional ligation methods with splint templates, then circularized probes can be formed, but various inefficiencies are introduced
Solution Approach 1:
The probe's stem region contains self-complementary sequences that automatically fold back to form hairpin structures, bringing the 5' and 3' ends into proximity without requiring external splint templates. This self-service mechanism eliminates the need for separate splint molecules, streamlining the ligation process and reducing optimization requirements.
Solution Approach 2:
The probe is pre-designed with self-complementary stem regions that automatically form hairpin structures under assay conditions, preliminarily positioning the ends for ligation before the actual circularization occurs. This preliminary structuring eliminates the need for complex splint-template assembly steps.
3Productivity
If the stem region is designed with self-complementary sequences, then ligation efficiency increases, but the probe structure becomes more complex
Solution Approach 1:
Only specific regions of the probe (the stem regions) are designed with self-complementary sequences, while other regions (target binding regions) maintain simple, functional sequences. This localized complexity approach improves ligation efficiency where needed without unnecessarily complicating the entire probe structure.
Solution Approach 2:
The self-complementary stem sequences are designed with specific length and GC content parameters that optimize hairpin formation and stability. By carefully controlling these parameters, the probe achieves high ligation efficiency while keeping the overall structure manageable through standardized design rules.
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
Enhances the efficiency of nucleic acid detection by increasing ligation efficiency and compaction of rolling circle amplification products, improving sensitivity and specificity in in situ analysis.
Implementation Method 1
the stem region is at one terminus of the first polynucleotide and comprises a second polynucleotide binding sequence and a self-complementary sequence
Implementation Method 2
the stem region brings a terminus of the first polynucleotide adjacent to a terminus of the second polynucleotide
Implementation Method 3
connecting the first polynucleotide and the second polynucleotide to generate a circularized probe at the location in the biological sample, wherein the connecting comprises: (i) ligating the first target binding region and the second target binding region using the target nucleic acid as a template, and (ii) ligating the stem region and the second probe region using the first polynucleotide as a template
Implementation Method 4
using a polymerase to perform rolling circle amplification (RCA) of the circularized probe, thereby generating a rolling circle amplification product (RCP)
Implementation Method 5
the first target binding region binds to a first target region in a target nucleic acid at a location in the biological sample and the second target binding region binds to a second target region in the target nucleic acid
Data Source
AI summary
The present disclosure relates, in some aspects, to methods and compositions for analyzing a biological sample. In some aspects, the methods comprise use of a probe set comprising a first and second polynucleotide which hybridize to a target nucleic acid in the biological sample and form a circularized probe. In some aspects, the methods and compositions provided herein improve the detection of nucleic acids in a biological sample.