Stimulus-Responsive Oligonucleotides for Single-Cell Sequencing
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Solution Overview
Problem
Next-generation sequencing technologies face challenges in distinguishing rare cell types from normal cells due to the masking of underlying heterogeneity in bulk cell populations, particularly in the analysis of somatic mutations and genomic changes at single cell resolution, where traditional library preparation methods can lead to background amplification and interference from de-activatable oligonucleotides.
Innovation Solution
The use of modifiable oligonucleotides, including de-activatable and activatable oligonucleotides, which can be selectively activated or deactivated by stimuli, allowing for controlled hybridization and primer extension in nucleic acid production, reducing background amplification and improving the specificity of library preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional library preparation methods are used, then the process is simple and straightforward, but background amplification and interference from de-activatable oligonucleotides occur
Solution Approach 1:
The patent employs dynamic oligonucleotides that can switch between active and inactive states in response to stimuli. De-activatable oligonucleotides are used during library preparation to prevent background amplification, then deactivated after use. Activatable oligonucleotides are activated only when needed for specific steps. This dynamic control resolves the contradiction by eliminating harmful background amplification while maintaining preparation simplicity through automated state changes.
Solution Approach 2:
The patent changes the functional state parameter of oligonucleotides from static to dynamic by incorporating stimulus-responsive elements. Chemical or physical stimuli trigger transitions between active and inactive states, controlling when oligonucleotides participate in reactions. This parameter change eliminates background amplification interference while maintaining ease of operation through automated state transitions.
2Reliability
If de-activatable oligonucleotides are used to reduce background amplification, then specificity improves, but the complexity of the system increases
Solution Approach 1:
The de-activatable and activatable oligonucleotides are designed to automatically respond to stimuli and change their own functional states without external intervention. The system uses self-service mechanisms where the oligonucleotides themselves undergo conformational changes or chemical modifications in response to environmental cues, eliminating the need for complex external control systems while maintaining high specificity.
Solution Approach 2:
The patent introduces stimulus-responsive intermediaries (such as chemical groups or structural elements) that mediate between the stimulus and the oligonucleotide function. These intermediaries translate physical or chemical stimuli into functional state changes, simplifying the overall system architecture by providing a straightforward mechanism for control without requiring complex regulatory systems.
3Object-affected harmful factors
If activatable oligonucleotides are used to control hybridization, then interference is minimized, but the difficulty of detecting and measuring increases
Solution Approach 1:
The patent incorporates stimulus-responsive elements that undergo detectable changes (such as conformational changes, fluorescence, or other signal changes) when activated or deactivated. These changes provide clear, measurable signals that indicate the functional state of the oligonucleotides, making detection and measurement straightforward while maintaining minimal interference during the library preparation process.
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 enables the effective synthesis of cDNA and reduces background amplification, enhancing the ability to analyze genomic and transcriptomic changes at single cell resolution by minimizing interference from unnecessary oligonucleotides, thereby improving the accuracy of sequencing results.
Implementation Method 1
controlled hybridization and primer extension in nucleic acid production
Data Source
AI summary
Provided are methods of producing product nucleic acids involving the use of oligonucleotides that are modified by the application of a stimulus. Aspects of such methods may include producing product nucleic acids using de-activatable oligonucleotides that are deactivated by a de-activating stimulus, as well as methods that may include producing product nucleic acids using activatable oligonucleotides that are activated by an activating stimulus and de-activatable oligonucleotides that are deactivated by a de-activating stimulus. Also provided are kits, compositions and devices that include de-activatable oligonucleotides or activatable and de-activatable oligonucleotides, e.g., for use in performing the methods as described herein.