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Labeling DNA Probes

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Updated: Nov 26, 2025

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Published on: October 18, 2024

Protocol for efficient fluorescence 3' end-labeling of native noncoding RNA domains.

Dahlia A Awwad¹, A Rachid Rahmouni², Fareed Aboul-Ela¹

¹Center of X-Ray Determination of Structure of Matter (CXDS), Helmy Institute of Medical Sciences, Zewail City of Science and Technology, Giza, Egypt.

|December 10, 2020

Summary

This study presents a streamlined protocol for synthesizing, purifying, and labeling structured noncoding RNAs (ncRNAs). It replaces hazardous radioactive methods with simpler fluorescence tagging, ensuring RNA integrity for downstream applications.

Keywords:

3′end-labeling Chemical tagging Cyanine hydrazides Fluorescence Native purification RNA in vitro transcription

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Last Updated: Nov 26, 2025

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

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Published on: October 18, 2024

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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Published on: August 6, 2014

Area of Science:

Molecular Biology
Biochemistry
RNA Biology

Background:

Noncoding RNAs (ncRNAs) are crucial regulators of biological processes.
Functional long ncRNAs (>200 nt) adopt specific secondary structures during transcription.
Maintaining native-like conditions is vital for structured ncRNA integrity during in vitro manipulation.

Purpose of the Study:

To develop an optimized protocol for obtaining homogenous, structured ncRNA samples.
To chemically tag the 3' termini of purified, structured ncRNAs (>200 nt).
To replace hazardous radioactive labeling with a safer, more efficient fluorescence tagging method.

Main Methods:

Co-transcriptional synthesis of structured ncRNAs.
Purification using centrifugal filtration to maintain homogeneity.
Chemical tagging of the 3' termini with fluorescence labels.

Main Results:

Achieved homogenous and mono-dispersed preparations of structured ncRNAs.
Successfully replaced hazardous radioactive labeling with fluorescence tagging.
Developed a simple, integrated protocol for synthesis, purification, and labeling.

Conclusions:

The protocol ensures structural integrity of ncRNAs throughout the process.
This method offers a safer and more efficient alternative to traditional RNA labeling techniques.
The versatile protocol is adaptable for various chemical tags and nucleic acid types (RNA/DNA).