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High-Sensitivity Fluorescence-Based Detection of Reverse Transcriptase Read-Through of GC-Rich Short Tandem Repeat

Weiqi Qiu¹, Catherine Hazard^1,2, Yujing Li³

¹Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States.

Analytical Chemistry

|February 13, 2025

Summary

Researchers developed a new method to prepare and detect GC-rich short tandem repeat (STR) RNAs, crucial for understanding STR expansion disorders. This advance improves the detection of disease-related RNA sequences.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Short tandem repeat (STR) RNAs are implicated in STR expansion-associated disorders.
GC-rich STR sequences, common in these disorders, present significant challenges for sample preparation and detection due to polymerase processivity issues.
Existing methods struggle with low yield and heterogeneity when amplifying GC-rich STR DNA.

Purpose of the Study:

To develop an efficient method for preparing and detecting GC-rich STR RNAs, specifically r(CGG)29 and r(G4C2)15, relevant to human FMR1 and C9ORF72 genes.
To establish a sensitive fluorescence-based detection platform for these challenging RNA sequences.
To provide structural insights into RNA-reverse transcriptase interactions during detection.

Main Methods:

In vitro preparation of high-yield and homogeneous GC-only STR RNAs (r(CGG)29 and r(G4C2)15).
Development of a fluorescence-based detection platform utilizing reverse transcriptases (RTases).
Analysis of RTase processing and structural insights into GC-rich STR RNAs.

Main Results:

Successful high-yield and homogeneous preparation of physiologically relevant GC-only STR RNAs.
A sensitive fluorescence-based detection platform capable of identifying read-through cDNA products with minimal RNA input.
Demonstration of the platform's versatile applications and structural insights into RNA-RTase interactions.

Conclusions:

The developed methods enhance the ability to characterize and target disease-relevant STR RNAs in vitro.
This work lays the foundation for directed evolution of RTases to improve detection of endogenous expanded GC-rich STR RNAs.
The findings offer a promising approach for studying STR expansion-associated disorders.