Strand-Displacement Imaging With Universal Adapters and 4-Way Exchange
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Solution Overview
Problem
Current DNA strand displacement methods for fluorescence microscopy are costly due to the need for conjugating distinct nucleic acid sequences to each target-recognition element, and suffer from inefficient strand displacement and non-specific binding issues.
Innovation Solution
A method involving target-recognizing antibodies bound to adapter strands, bridge strands, and imager strands labeled with detectable labels, utilizing shorter DNA sequences and 4-way branch migration to introduce and remove imager strands, along with blocker and bouncer strands for multiplexed imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If distinct nucleic acid sequences are conjugated to each target-recognition element, then specific target recognition is achieved, but cost and complexity increase
Solution Approach 1:
The patent employs universal adapter strands that can bind to multiple different target-recognition elements through a common interface, eliminating the need for distinct nucleic acid sequences for each target. The adapter strand serves as a universal intermediary that maintains specific binding while reducing conjugation complexity across multiple targets.
Solution Approach 2:
The adapter strand acts as an intermediary component between the target-recognition element and the imaging reagent. This mediator allows for flexible, specific targeting without requiring direct conjugation between each target and its imaging reagent, thereby reducing overall system complexity and cost.
2Stability of the object's composition
If longer DNA strands are used for strand displacement, then binding stability is improved, but non-specific binding increases
Solution Approach 1:
The patent designs the adapter strand with a specific local structure including a toehold domain and a binding domain. The toehold domain provides a controlled interface for strand displacement while the binding domain ensures specific target recognition. This localized functional design achieves stable binding without requiring excessively long DNA strands that would cause non-specific binding.
3Object-generated harmful factors
If shorter DNA sequences are used, then non-specific binding is reduced, but strand displacement efficiency decreases
Solution Approach 1:
The adapter strand is segmented into distinct functional regions: a toehold domain that facilitates efficient strand displacement through controlled hybridization, and a binding domain that provides specific target recognition. This segmentation allows shorter overall sequences to achieve both efficient displacement and specific binding without non-specific interactions.
4Adaptability or versatility
If multiple imaging rounds are performed sequentially, then multiplexed imaging is achieved, but time consumption increases
Solution Approach 1:
The patent enables continuous multiplexed imaging by maintaining the adapter strand-bound target-recognition elements in the sample and sequentially introducing different imaging reagents that bind to the adapter strand. This continuous approach allows multiple targets to be imaged in sequence without removing or replacing the initial adapter-strand complexes, reducing overall time compared to separate imaging assays.
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 reduces non-specific binding and costs by using universal adaptors and shorter DNA sequences, enabling efficient and cost-effective multiplexed imaging with reduced cross-reactivity.
Implementation Method 1
providing an imager strand oligonucleotide that hybridizes to the docking strand oligonucleotide at least in the region of the docking strand that does not hybridize to the blocker strand
Implementation Method 2
allowing the imager strand to hybridize to the docking strand, displacing the blocker strand
Implementation Method 3
providing a bridge strand oligonucleotide with a region capable of specifically binding the adapter strand oligonucleotide
Implementation Method 4
providing at least one target-recognizing antibody bound to an adapter strand oligonucleotide
Data Source
AI summary
The present disclosure describes various improved methods for imaging at least one target in a sample, including methods employing an adapter strand oligonucleotide and a bridge strand oligonucleotide. Some methods also employ bouncer oligonucleotides and/or blocker oligonucleotides. Some methods also use two partial docking strands to detect proximity of the partial docking strands to each other.