Fluorescent Base Analogues for Nucleic Acid Characterization
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Solution Overview
Problem
Current methods for detecting nucleic acid structure and interactions rely on extrinsic fluorescent probes that alter the mobility and interactions of nucleic acid molecules, leading to inaccurate reflections of their behavior.
Innovation Solution
The use of fluorescent base analogues as intrinsic probes, which are designed as structural analogs to natural bases, minimizing perturbation and providing high sensitivity to conformational changes through fluorescence properties such as intensity, anisotropy, and energy transfer characteristics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If extrinsic fluorescent probes are used to detect nucleic acid structure and interactions, then detection capability is improved, but the probes alter the mobility and interactions of nucleic acid molecules leading to inaccurate reflections of their behavior
Solution Approach 1:
The nucleic acid molecule itself serves as the fluorescent probe through incorporation of fluorescent base analogues, eliminating the need for separate extrinsic probes. This self-service approach allows the molecule to report its own structural and interactional state without external interference, resolving the contradiction between detection capability and behavioral accuracy
Solution Approach 2:
The invention changes the chemical parameter of the nucleic acid by incorporating fluorescent base analogues that modify the molecule's intrinsic properties. These analogues alter fluorescence characteristics while maintaining minimal perturbation to the molecule's natural behavior, enabling accurate detection without the drawbacks of bulky extrinsic probes
2Illumination intensity
If bulky fluorescent labels and linkers are attached to nucleic acid molecules, then fluorescence detection is enabled, but the labels alter the mobility and interactions of the nucleic acid through chemical interactions and steric hindrance
Solution Approach 1:
The fluorescent base analogues are incorporated at specific local positions within the nucleic acid sequence, allowing fluorescence detection at targeted locations without requiring bulky labels throughout the molecule. This local quality approach enables detection while minimizing overall steric hindrance and chemical interference
Solution Approach 2:
The invention changes the fundamental parameter of fluorescence generation from extrinsic labeling to intrinsic base analogue incorporation. This parameter change eliminates the need for bulky linkers and large fluorescent labels, reducing steric hindrance and chemical interactions while maintaining detectable fluorescence signals
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
Enables reliable, real-time characterization of nucleic acid molecules' structure and interactions by transforming them into intrinsic fluorescent probes, providing accurate data on conformational changes and interactions with other molecules.
Implementation Method 1
measuring the fluorescent properties, such as the fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer characteristics produced by the incorporated fluorescent nucleotide(s)
Implementation Method 2
FlRET occurs when the two fluorophores are brought into proximity under exciting radiation. The direct correlation of measurable FRET efficiency with the distance between the two chromophores
Data Source
AI summary
This invention provides methods, apparatus and kits for the quantitative and qualitative characterization of the nucleic acid molecule's behavior by modify the nucleic acid molecules to incorporate selected fluorescent base analogues (FBAs). The methods generally place one or more fluorescent base analogue into a nucleic acid molecule (e.g., an oligonucleotide) to replace the corresponding normal base(s), arrange fluorescent base analogues as intrinsic fluorescent probes by using direct excitation, indirect excitation, and excimer emission labeling schemes, introducing so modified nucleic acid molecules into the matrix with interested condition and measuring the fluorescent properties of the modified nucleic acid molecules at the specific emission wavelength of FBA(s). The apparatus is designed to irradiate the FBA(s) incorporated nucleic acid molecule at a wavelength in the range of 240 nm-280 nm and detect the fluorescent activities at the specific emission wavelength of the respective FBA(s). The kit provides oligonucleotides modified by multiple FBAs in the position of critical portions. It utilizes simultaneous indirect excitation labeling scheme for qualitative and quantitative investigation of nucleic acid molecules' interaction in vitro and in vivo.