Engineered Surfaces for Single-Base DNA and RNA Modification Profiling
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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 multiplexed detection, leading to inconsistent and low-resolution identification of modifications, which hinders understanding of their role in health and disease.
Innovation Solution
Compositions and methods utilizing substrates with binding domains and adapters that specifically recognize non-canonical features of nucleic acids, coupled with nucleic acid barcode sequences, enable high-throughput, sensitive, and accurate profiling of multiple modifications simultaneously.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If chemical derivatization methods and molecular recognition are used for profiling DNA and RNA modifications, then detection capability is improved, but sensitivity and measurement precision deteriorate
Solution Approach 1:
The patent introduces an intermediary molecular recognition system consisting of engineered proteins with high-affinity binding domains that specifically recognize modified nucleotides. These binding domains act as mediators between the modified nucleic acids and the detection system, enabling sensitive and specific detection through affinity-based enrichment followed by sequencing. This intermediary approach overcomes the limitations of direct chemical derivatization methods by providing enhanced binding specificity and sensitivity.
2Reliability
If existing methods are used for identifying modifications, then some detection is achieved, but single-base resolution and location precision are insufficient
Solution Approach 1:
The patent segments the detection process into distinct functional modules: (1) affinity enrichment using modified nucleotide-specific binding domains, (2) adapter ligation for molecular tagging, and (3) high-throughput sequencing for precise localization. This segmentation allows each module to optimize for its specific function, with the binding domains providing modification-specific enrichment and the sequencing technology providing single-base resolution mapping of modification locations throughout the genome.
3Adaptability or versatility
If current profiling methods are applied, then limited modifications can be detected, but multiplexed detection capability and versatility are limited
Solution Approach 1:
The patent creates a universal platform for detecting multiple types of DNA and RNA modifications simultaneously. The system uses a common molecular recognition framework where different binding domains can be exchanged to target various modified nucleotides (such as 5mC, 5hmC, 6mA, etc.). This universal approach allows multiplexed detection of numerous different modifications using the same overall methodology, greatly enhancing versatility and the number of modifications that can be profiled in a single experiment.
4Difficulty of detecting and measuring
If chemical derivatization and molecular recognition methods are used, then some profiling is achieved, but nucleic acid degradation and fragmentation occur
Solution Approach 1:
The patent replaces harsh chemical derivatization methods with a gentler molecular recognition-based approach using engineered protein binding domains. Instead of using chemical reactions that can damage nucleic acids, the system uses specific protein-nucleic acid interactions that occur under physiological conditions. This substitution of chemical mechanisms with biological recognition mechanisms preserves nucleic acid integrity while achieving effective profiling.
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
Provides highly parallelized, sensitive, and accurate methods for identifying and locating DNA and RNA modifications with single-base resolution, enabling comprehensive analysis of modification distribution and function.
Implementation Method 1
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 identification and/or 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.