Riboswitch Conformational Detection via Fluorescent Probes

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Solution Overview

Problem

There is a critical need to understand the folding routes, real-time conformational transition states, and switching mechanisms of riboswitches to develop RNA-based drug targets for controlling gene expression, as current efforts have not adequately addressed these aspects.

Innovation Solution

A method involving the use of chemically modified ligands to detect riboswitches in biological samples through fluorescence or absorbance, utilizing aptamer domains and optical detection techniques to identify and control the conformational states of riboswitches, allowing for the regulation of gene expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If riboswitches are used to control gene expression, then gene regulation capability is improved, but understanding of folding routes and conformational transition states remains insufficient

Engineering Contradiction:

Improvegene regulation capabilityVSAvoidfolding route information

Core Design Contradiction:

ReliabilityVSLoss of information

Solution Approach 1:

The patent introduces fluorescent probes as intermediary molecules that bind to riboswitches and report their conformational states. These probes act as mediators between the riboswitch structure and detection methods, enabling indirect observation of folding routes and transition states that would otherwise be inaccessible.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent utilizes fluorescent probes that undergo color/fluorescence changes upon binding to specific riboswitch conformations. This allows visual detection and differentiation of various folding states and transition states, transforming invisible conformational changes into detectable optical signals.

Inventive Principle:

Principle #32Color changes

2Measurement precision

If fluorescent probes are used to detect riboswitch conformational states, then detection capability is improved, but complexity of the detection system increases

Engineering Contradiction:

Improveconformational state detectionVSAvoiddetection system complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The fluorescent probes are designed to self-report the conformational states of riboswitches through their intrinsic fluorescence properties. The probes automatically bind to specific states and emit signals without requiring external manipulation or complex instrumentation, simplifying the overall detection approach.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The patent replaces complex mechanical or structural analysis methods with optical detection using fluorescent probes. Instead of requiring sophisticated mechanical instruments to directly observe riboswitch conformations, the system uses optical signals that are easier to detect and interpret.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Loss of information

If real-time monitoring of riboswitch folding is implemented, then dynamic behavior understanding is improved, but experimental complexity increases

Engineering Contradiction:

Improvedynamic behavior informationVSAvoidexperimental complexity

Core Design Contradiction:

Loss of informationVSDevice complexity

Solution Approach 1:

The fluorescent probes enable continuous, real-time monitoring of riboswitch folding dynamics without disrupting the natural process. The probes remain bound to the riboswitch throughout the folding transition, providing uninterrupted information about conformational changes from initial states to final structures.

Inventive Principle:

Principle #20Continuity of useful action

Solution Approach 2:

The probes serve as intermediaries that penetrate into the dynamic folding process without interfering with it. They report conformational changes in real-time while maintaining their own structural integrity and binding affinity, allowing continuous observation without experimental intervention.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 the detection and regulation of riboswitches, providing insights into RNA-mediated gene regulation and potential RNA-based therapeutic targets for controlling gene expression.

Implementation Method 1

detecting fluorescence of the chemically modified ligand bound to the biological molecule comprising one or more riboswitch

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

a binding of the chemically modified ligand to the biological molecule comprising one or more riboswitch is required for optical detection of the biological molecule comprising one or more riboswitch by fluorescence or absorbance

Methodology Applied

Scientific EffectAbsorbance: Absorption (EM radiation)

Data Source

PatentUS20240384342A1Materials and methods for controlling gene expression

Publication Date: 2024.11.21 MANDAL MAUMITA

AI summary

A high-resolution dual-beam counter propagating optical-tweezers instrument was designed that can measure forces at <1 pN and one nanometer distance at a temporal resolution of 25 μs with high accuracy and precision. Using the high-resolution optical-tweezers, time-dependent conformational switching and structural rearrangements in a single-molecule of the guanine aptamer were identified that follow a modified induced-fit model, where guanine remodels multiple barriers and triggers the receptor conformation rapidly to synchronize with the elongating transcriptional machinery for controlling gene regulation.