Nucleic Acid Beacons for Fluorescent In-Situ Hybridisation
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Solution Overview
Problem
Current molecular beacon designs for in-situ hybridization and chip technology face challenges such as poor solubility of PNA-based oligonucleotides, stringent wash steps, and difficulty in achieving standardized hybridization conditions for detecting microorganisms, particularly bacteria, due to sterical hindrance and thermodynamic incompatibilities with FRET requirements.
Innovation Solution
Development of nucleic acid beacons capable of forming a stem-loop structure with a complementary sequence and an effector-inhibitor pair, where the inhibitor prevents effector activity when the beacon forms a stem, allowing hybridization under conditions where the stem is open, and using Mg2+ buffers to induce stem formation post-hybridization, ensuring successful hybridization with rRNA targets without sterical limitations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PNA-based molecular beacons are used for in-situ hybridization, then hybridization sensitivity is improved, but solubility of the oligonucleotides deteriorates
Solution Approach 1:
The patent modifies the chemical composition parameters of the beacon molecule by incorporating DNA bases (adenine, thymine, guanine, cytosine) alongside PNA bases, and adjusting the stem-loop structure parameters, to achieve optimal solubility while preserving hybridization sensitivity
Solution Approach 2:
The patent creates a composite oligonucleotide structure combining PNA and DNA components, where the DNA portions provide solubility while the PNA portions maintain hybridization sensitivity, resolving the contradiction between these two properties
2Measurement precision
If stringent wash steps are applied to remove unbound probes, then detection specificity is improved, but procedure complexity and time consumption increase
Solution Approach 1:
The patent designs the beacon with a stem-loop structure where the inhibitor is pre-positioned to automatically quench the effector when the beacon is unbound, eliminating the need for separate wash steps to achieve specificity
Solution Approach 2:
The beacon molecule performs self-regulation through its stem-loop structure, automatically activating or deactivating the effector based on hybridization state without requiring external wash interventions, thereby simplifying the procedure
3Productivity
If multiple probes are designed to work simultaneously under identical conditions, then productivity is improved, but standardization of probe characteristics becomes more difficult
Solution Approach 1:
The patent establishes universal design parameters for the beacon structure including standardized stem-loop configurations, effector-inhibitor pairs, and hybridization conditions that enable multiple different probes to function simultaneously under identical conditions across various applications
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 designed beacons achieve successful hybridization with microorganisms under standardized conditions, reducing the need for complex wash steps and enabling simultaneous use of multiple probes on a single chip, improving the efficiency and reproducibility of microorganism detection in clinical and routine microbiological applications.
Implementation Method 1
a pair of two complementary sequences (a2) capable of forming a stem
Implementation Method 2
complementary sequences capable of forming a stem
Implementation Method 3
capable of forming a hybrid with a target nucleic acid sequence
Implementation Method 4
sequence (a1) comprising a sequence complementary to the target nucleic acid sequence
Implementation Method 5
using Mg2+ buffers to induce stem formation post-hybridization
Data Source
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AI summary
The present invention relates to beacons for fluorescent in-situ hybridisation and chip technology.