Barcoded Nucleic Acid Profiling for Multiplexed DNA and RNA Modifications
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Solution Overview
Problem
Current methods for profiling epitranscriptomic and epigenetic modifications in nucleic acids lack sensitivity, specificity, and are not amenable to multiplexing, leading to inconsistent results and inability to identify modifications at single-base resolution.
Innovation Solution
Development of nucleic acid-binding molecules with specific binding domains and adapters that allow for highly parallelized, sensitive, and accurate profiling of DNA and RNA modifications by transferring unique barcodes to target nucleic acids, enabling simultaneous identification and localization of multiple modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing profiling methods (chemical derivatization + molecular recognition + sequencing by reverse transcription) are used, then a limited number of DNA and RNA modifications can be profiled, but the methods lack high sensitivity, cause nucleic acid degradation/fragmentation, cannot identify modification locations at single-base resolution, and are not amenable to multiplexing
Solution Approach 1:
The patent divides the profiling task into separate detection channels, where each modification type is detected by a specific nucleic acid-binding molecule (e.g., antibody, aptamer, or protein domain). This segmentation allows simultaneous multiplexed detection of multiple modification types without interference, resolving the contradiction between profiling capacity and detection precision.
Solution Approach 2:
The patent introduces nucleic acid-binding molecules as intermediaries that specifically recognize and bind to different modification types. These binding molecules serve as mediators between the modifications and the detection system, enabling sensitive and specific identification of multiple modifications simultaneously without degrading the nucleic acids.
2Reliability
If existing profiling methods are used, then some modifications can be detected, but the methods cause nucleic acid degradation/fragmentation and give conflicting findings
Solution Approach 1:
The patent employs nucleic acid-binding molecules that perform detection without altering or degrading the target nucleic acids. The binding molecules merely recognize and bind to modifications, leaving the nucleic acid structure intact for subsequent analysis, thereby eliminating the harmful degradation effect while maintaining detection reliability.
3Productivity
If existing methods are used, then a limited number of modifications can be profiled, but the methods are not amenable to multiplexing
Solution Approach 1:
The patent creates a universal detection platform where multiple types of nucleic acid-binding molecules can be used simultaneously to detect different modification types. The common workflow (binding → sequencing by ligation) remains the same, allowing multiplexed profiling of numerous modifications without proportionally increasing system complexity.
4Measurement precision
If existing methods are used, then modifications can be detected, but the methods lack specificity and cannot locate modifications at single-base resolution
Solution Approach 1:
The patent adds a dimensional approach by using nucleic acid-binding molecules that can be coupled with various technologies (sequencing by ligation, nanopore sequencing, etc.). This dimensional expansion enables precise location of modifications at single-base resolution while maintaining operational accessibility through established sequencing platforms.
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 simultaneous, high-throughput analysis of a potentially unlimited number of DNA and RNA modifications at single-molecule level, providing precise identification and localization of non-canonical features with high sensitivity and accuracy.
Implementation Method 1
molecular recognition (typically using antibodies, both for enrichment and detection)
Implementation Method 2
Combinations of chemical derivatization methods, molecular recognition (typically using antibodies, both for enrichment and detection), and sequencing by reverse transcription
Implementation Method 3
molecular recognition (typically using antibodies, both for enrichment and detection)
Data Source
AI summary
Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and/or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA/RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA/RNA modifications in parallel.