Nucleic Acid Assembly pH Sensing via i-Motif Conformational Switching
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Solution Overview
Problem
Current methods for obtaining high-resolution spatiotemporal chemical maps of second messengers within cells, particularly pH, are limited by imaging artifacts and temporal resolution, and DNA-based sensors face challenges in delivering precise intracellular targeting and response times.
Innovation Solution
A nucleic acid assembly (NAA) comprising a sensor domain and handle domain, conjugated with an assembly interfaceable motif (AIM) through an artificial receptor, enables precise intracellular targeting and rapid pH sensing by forming an i-motif at acidic pH, with a recombinant antibody binding to the handle domain for trafficking and localization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional pH sensing methods (fluorescent probes, CARS, SIMS, MALDI) are used to obtain chemical maps of second messengers, then spatial resolution can be achieved, but temporal resolution deteriorates and imaging artifacts increase
Solution Approach 1:
The patent replaces conventional imaging-based pH sensing methods (fluorescence microscopy, CARS, SIMS, MALDI) with a DNA-based nanosensor system. The DNA sensor utilizes conformational changes (i-motif formation) in response to pH changes, which are then detected through fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores attached to the DNA structure. This substitution enables both high spatial resolution (nanoscale sensing) and improved temporal resolution (real-time monitoring) while avoiding imaging artifacts associated with conventional methods.
2Measurement precision
If DNA-based sensors are used for pH sensing in living cells, then spatial resolution is improved, but delivery to precise intracellular locations and response time are limited
Solution Approach 1:
The DNA sensor is divided into distinct functional domains: a pH-sensing domain that forms an i-motif structure in response to acidic pH, a handle domain for conjugation with targeting motifs, and fluorophore attachment sites for FRET-based detection. This segmentation allows each domain to be optimized independently - the sensing domain for rapid conformational response, the handle domain for efficient cellular delivery, and the fluorophores for sensitive detection - thereby improving both response time and spatial resolution simultaneously.
Solution Approach 2:
The patent optimizes the DNA sequence parameters to enhance sensing speed and delivery efficiency. The i-motif forming sequence is designed with specific cytosine repeat patterns that enable rapid conformational switching in response to pH changes. The handle domain sequence is optimized for conjugation with cell-penetrating peptides or organelle-specific targeting motifs, accelerating delivery to intracellular locations. These parameter optimizations resolve the contradiction between spatial resolution and response time.
3Ease of operation
If fluorescent pH probes are conjugated to endocytosable ligands for intracellular targeting, then delivery to specific compartments is improved, but ligand structure or trafficking is disrupted
Solution Approach 1:
The patent introduces a DNA handle domain as an intermediary between the pH-sensing i-motif structure and the cell-penetrating or targeting ligands. The handle domain serves as a modular connection point that allows ligand attachment without interfering with the pH-sensing function of the i-motif. This intermediary structure enables flexible conjugation of various targeting motifs (e.g., cell-penetrating peptides, organelle-specific sequences) while maintaining the natural trafficking behavior of the ligands and preserving their ability to deliver the sensor to specific intracellular compartments.
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
The NAA achieves fast response times and accurate pH mapping across various cellular compartments, overcoming previous limitations in DNA sensor delivery and response times, allowing for detailed spatiotemporal chemical mapping within living cells.
Implementation Method 1
Acidic pH causes the formation of a non-Watson-Crick based DNA motif called the I-tetraplex, or i-motif that is then transduced into a large scale conformational change of the overall DNA assembly
Implementation Method 2
a recombinant antibody binding to the handle domain for trafficking and localization
Implementation Method 3
The NAA achieves fast response times and accurate pH mapping across various cellular compartments
Data Source
Figure 1a~1g
Figure 2~3b
Figure 4~5c
AI summary
The present disclosure relates to a nucleic acid assembly (NAA), comprising sensor domain and handle domain; an assembly interfaceable motif (AIM) sequence optionally along with intracellular targeting motif (ITM) sequence; and an AIM- NAA complex. It also relates to a vector comprising assembly interfaceable motif sequence optionally along with intracellular targeting motif sequence and a cell comprising the vector. Further, the instant disclosure also provides a method to obtain the nucleic acid assembly, method of intracellular targeting and kit thereof.