RNA Probe Ligation Barcoding Without Reverse Transcription
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for analyzing nucleic acids, particularly RNA, often require reverse transcription and lack efficient methods for controlled analysis and processing of biological particles, nucleic acids, and proteins within partitions such as droplets or wells.
Innovation Solution
A method involving hybridization of probes to target regions of nucleic acid molecules, linking them to form probe-linked nucleic acids, and barcoding these molecules within partitions, which can be droplets or wells, without the need for reverse transcription, using reactive moieties and nucleic acid barcode molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If reverse transcription is used for RNA analysis, then RNA can be converted to DNA for sequencing, but the process becomes more complex and requires additional enzymes and conditions
Solution Approach 1:
The invention extracts and eliminates the reverse transcription step from the traditional RNA analysis workflow. By using RNA-specific probes that directly hybridize to RNA targets and incorporating RNA-compatible barcode molecules, the method removes the need for reverse transcription while maintaining the ability to analyze RNA samples through sequencing.
Solution Approach 2:
Instead of converting RNA to DNA through reverse transcription (the conventional approach), the invention inverts the approach by designing a system that works directly with RNA molecules. RNA probes hybridize to RNA targets, and RNA barcodes are incorporated directly into the RNA molecule, allowing RNA analysis without conversion to DNA.
2Measurement precision
If probes are hybridized to target regions and linked to form probe-linked nucleic acids, then specific nucleic acid sequences can be targeted and analyzed, but the ligation step requires precise conditions and may reduce yield
Solution Approach 1:
The invention modifies the ligation chemistry by using reactive moieties such as azide and alkyne groups that undergo copper-free click chemistry or other bioorthogonal reactions. These parameter changes in the chemical reaction conditions allow ligation to proceed under more physiological and less stringent conditions, improving both efficiency and yield while maintaining target specificity.
Solution Approach 2:
The invention introduces reactive moieties as intermediary functional groups on the probes that facilitate ligation. These reactive groups (azide, alkyne, or other bioorthogonal handles) act as mediators that enable efficient joining of probes to the nucleic acid target without requiring traditional phosphodiester bond formation, thereby improving ligation efficiency.
3Measurement precision
If barcoding is performed within partitions such as droplets or wells, then controlled analysis of individual nucleic acid molecules is enabled, but partitioning and handling become more complex
Solution Approach 1:
The invention segments the analysis process by performing barcoding and initial amplification steps within individual partitions (droplets or wells), ensuring that each nucleic acid molecule is independently labeled. This segmentation enables precise tracking of individual molecules while the subsequent pooling and bulk amplification steps simplify handling by processing many partitions simultaneously.
Solution Approach 2:
The invention performs the barcoding step as a preliminary action before bulk amplification. By incorporating unique barcodes onto each nucleic acid molecule while still in partitioned form, the method enables subsequent pooling and processing of all samples together through the amplification and sequencing steps, thereby simplifying overall operation while maintaining single-molecule resolution.
4Length of stationary object
If multiple probes are hybridized to adjacent target regions and linked, then longer contiguous sequences can be assembled, but the probability of successful ligation decreases with distance between targets
Solution Approach 1:
The invention changes the chemical parameters of the ligation reaction by using bioorthogonal chemistry (click chemistry, strain-promoted azide-alkyne cycloaddition, or other copper-free methods). These parameter changes allow ligation to occur efficiently even when target regions are farther apart, as the reactive moieties can tolerate greater flexibility and distance compared to traditional enzymatic ligation, thereby enabling assembly of longer contiguous sequences with maintained reliability.
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 efficient and controlled analysis of nucleic acids by obviating the need for reverse transcription and allowing for amplification and sequencing of barcoded nucleic acid molecules within partitions, facilitating accurate and efficient processing of biological samples.
Implementation Method 1
hybridize the first probe sequence of the first probe to the first target region of the nucleic acid molecule
Implementation Method 2
hybridize the third probe sequence of the second probe to the second target region of the nucleic acid molecule
Implementation Method 3
subjecting the first reactive moiety and the second reactive moiety to conditions sufficient to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.