Nucleic Acid Lysosome Sensors for Simultaneous pH and Ion Mapping
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Solution Overview
Problem
Current methods are unable to chemically resolve lysosome populations in live cells, as they lack the capability to quantify Ca²⁺ and Cl⁻ concentrations within lysosomes, which are critical for understanding lysosome function and dysfunction in diseases like Alzheimer's and Parkinson's.
Innovation Solution
The use of nucleic acid complexes, such as CalipHluor and ChloropHore, which include a Ca²⁺ or Cl⁻ fluorophore crosslinked to a single-stranded nucleic acid molecule, allowing simultaneous determination of pH and Ca²⁺ or Cl⁻ concentration through signal intensity measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional imaging methods (electron microscopy, bright field imaging) are used to distinguish lysosome populations, then morphological classification is possible, but chemical resolution and quantification of ion concentrations (Ca²⁺, Cl⁻) and pH cannot be achieved
Solution Approach 1:
The patent combines multiple sensing capabilities (pH sensing, Ca²⁺ sensing, and Cl⁻ sensing) into a single integrated nucleic acid complex. The complex contains a pH-sensitive fluorophore, a Ca²⁺-sensitive fluorophore (Rhod-5F), and a Cl⁻-sensitive fluorophore (BAC), all linked to a complementary DNA strand that forms an i-motif structure. This merging allows simultaneous measurement of all three parameters in live cells, achieving chemical resolution of lysosome populations without requiring multiple separate measurement systems.
Solution Approach 2:
The nucleic acid complex serves multiple functions simultaneously: it acts as a pH sensor, a Ca²⁺ sensor, and a Cl⁻ sensor. The complementary DNA strand forms an i-motif structure that responds to pH changes, while attached fluorophores detect Ca²⁺ and Cl⁻ concentrations. This multi-functionality enables comprehensive chemical characterization of lysosome populations in a single measurement, resolving the limitation of conventional methods that could only provide morphological classification.
2Loss of information
If lysosomes are assayed as a single population, then general lysosomal function can be monitored, but sub-population-specific functions and mechanisms cannot be distinguished
Solution Approach 1:
The patent utilizes fluorescence emission at different wavelengths to distinguish various ion concentrations and pH levels across lysosome sub-populations. The pH-sensitive fluorophore emits at different wavelengths based on pH, Rhod-5F emits differently based on Ca²⁺ concentration, and BAC emits differently based on Cl⁻ concentration. By measuring the intensity ratios of these emissions, the system can identify and characterize distinct lysosome sub-populations (such as LAMP1+ vs LAMP2+ lysosomes) with their specific chemical environments, preventing loss of information about sub-population functions.
Solution Approach 2:
The patent creates a chemical copy or representation of the lysosomal environment through the nucleic acid complex's response. The i-motif forming DNA strand copies the pH condition through its structural change, while the fluorophores copy the ion concentrations through their emission intensities. This allows the complex to serve as a molecular reporter that replicates the chemical state of different lysosome sub-populations, enabling differentiation without requiring physical separation or reducing analysis efficiency.
3Measurement precision
If electrophysiology methods are used to discover ion channels, then channel discovery is possible, but quantification of intracellular ion concentrations in live cells cannot be achieved
Solution Approach 1:
The nucleic acid complex is designed to be self-contained and self-reporting. The complementary DNA strand automatically forms the i-motif structure in response to pH changes within the lysosome, and the fluorophores automatically report ion concentrations through their emission properties. The complex enters live cells and performs measurements autonomously without requiring external electrophysiological equipment or complex experimental setups, making intracellular ion concentration quantification as easy as imaging the fluorescence signal.
Solution Approach 2:
The patent replaces the mechanical/electrical measurement system of electrophysiology with an optical measurement system based on fluorescence. Instead of using electrodes and electrical signals to measure ion concentrations, the system uses light-emitting fluorophores whose emission intensities and wavelengths change in response to ion concentrations and pH. This substitution enables non-invasive, easy-to-perform measurements in live cells while achieving precise quantification of intracellular ion concentrations.
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
These complexes enable precise, single-endosome resolution of lysosomal pH and ion concentrations, facilitating the identification of lysosome-specific mechanisms and potential drug targets for lysosomal storage diseases.
Implementation Method 1
a Ca²⁺ or Cl⁻ fluorophore crosslinked to a single-stranded nucleic acid molecule, allowing simultaneous determination of pH and Ca²⁺ or Cl⁻ concentration through signal intensity measurement
Implementation Method 2
pH-induced FRET between Alexa488 (donor, D, sphere) and Alexa647 (acceptor, A, star) reports on pH ratiometrically
Data Source
Figure 1A
Figure 1B~1D
Figure 2A~2B
AI summary
This disclosure relates to methods for determining pH and also calcium (Ca2+) concentration or chloride (Cl-) concentration in biological samples. More particularly, this disclosure relates to methods capable of simultaneously determining pH and Ca2+ concentration, or pH and Cl- concentration using nucleic acid complexes.