2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Ning Zhang; Shundi Shi; Barney Yoo; Xiaohong Yuan; Wenjia Li; Shenglong Zhang

doi:10.3791/61281

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures wi...

JoVE Journal
Genetics

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Genetics

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Full Text

2,376 Views

05:41 min

July 10, 2020

DOI: 10.3791/61281-v

Ning Zhang^1,2, Shundi Shi², Barney Yoo³, Xiaohong Yuan¹, Wenjia Li⁴, Shenglong Zhang¹

¹Department of Biological and Chemical Sciences,New York Institute of Technology, ²Department of Chemical Engineering,Columbia University, ³Department of Chemistry, Hunter College,City University of New York, ⁴Department of Computer Science,New York Institute of Technology

Overview

This article presents a novel LC-MS-based sequencing method for short RNA that eliminates the need for a cDNA intermediate. The protocol allows for the simultaneous sequencing of various RNA modifications with single-base precision.

Key Study Components

Area of Science

RNA sequencing
Epitranscriptomics
Biotechnology

Background

Current RNA sequencing methods often rely on cDNA intermediates.
Enzymatic errors can affect sequencing accuracy.
Simultaneous sequencing of RNA modifications is challenging.
Understanding RNA modifications is crucial for studying human diseases.

Purpose of Study

To introduce a direct sequencing method for RNA.
To enable the study of multiple RNA modifications in one analysis.
To provide a reliable tool for epitranscriptomic research.

Methods Used

LC-MS-based sequencing technique.
Direct sequencing of single-stranded, mixed, or modified RNA.
Elimination of cDNA synthesis step.
Workflow designed for high precision and accuracy.

Main Results

The method successfully sequences short RNA (<35 nt) without cDNA.
Simultaneous detection of various RNA modifications was achieved.
The technique demonstrated resilience against enzymatic errors.
Potential applications in diagnostics for human diseases were identified.

Conclusions

This new sequencing method could become a standard for modified RNA analysis.
It offers a comprehensive approach to studying RNA modifications.
The protocol has implications for future research in RNA biology.

Frequently Asked Questions

What is the main advantage of this new sequencing method?

The main advantage is the ability to sequence RNA directly without the need for cDNA, allowing for higher accuracy and the simultaneous analysis of multiple modifications.

Can this method be used for all types of RNA?

Yes, it is applicable to single-stranded, mixed, or modified RNA samples.

How does this method compare to traditional RNA sequencing?

Unlike traditional methods, this approach does not rely on enzymatic processes that can introduce errors, making it more reliable for precise sequencing.

What are the potential applications of this technology?

It can be developed into a diagnostic tool for identifying RNA segments related to human diseases and for studying RNA modifications at the epitranscriptomic level.

Is this method suitable for clinical use?

While it shows promise, further validation and standardization are needed before it can be widely adopted in clinical settings.

What is the significance of studying RNA modifications?

Studying RNA modifications is crucial for understanding their roles in gene regulation and their implications in various diseases.

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (<35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

This protocol covers all essential steps needed when using the new method developed to directly sequencing RNA, regardless if the RNA sample is single-stranded, mixed, or modified. The method is not affected by enzymatic error or base complementarity. It provides a direct workflow and general solution that makes it possible to sequence different RNA modifications simultaneously rather than one specific modification at a time.

This technology can be developed into a diagnostic tool to determine signature RNA segments to relate it to human disease and an accurate tool to study RNA modifications at the epitranscriptomic level. The method does not rely on any previous sequencing method. Instead, it is a completely new approach, and we aim to make it to the go-to method for sequencing any modified RNA.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

2D-HELS MS Seq LC-MS RNA Sequencing Nucleotide Modifications De Novo Sequencing Diagnostic Tool Epitranscriptomic Level T4 RNA Ligase MassHunter Software RNA Purification Compound Extraction Visual Demonstration RNA Sample Preparation