Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems
This study introduces a hybridization-based purification method
that enables high-yield production of native and engineered tRNAs
from E. coli, preserving their natural post-transcriptional modifications and
translational activity. The method outperforms traditional in vitro transcription,
providing the first complete modification profiles of key tRNAs and offering a scalable
toolkit for translation and synthetic biology research.
New insights into the pathogenesis of SARS-CoV-2 during and after the COVID-19 pandemic
This review provides a comprehensive overview of SARS-CoV-2
(COVID-19), detailing its multisystem impact on the respiratory,
renal, gastrointestinal, and nervous systems, as well as the risk
factors influencing disease severity. It emphasizes the virus's interaction with
the ACE2 receptor, the resulting pulmonary complications, and the
long-term post-COVID sequelae, highlighting the need for deeper understanding
of its pathophysiology, immune response, and long-term health consequences.
Mass Spectrometry-Based Direct Sequencing of tRNAs De Novo and Quantitative Mapping of Multiple RNA Modifications
This study presents MLC-Seq, a mass-spectrometry ladder
complementation sequencing method that enables de novo,
full-length sequencing of heterogeneous tRNAs with
single-nucleotide precision. Unlike next-generation sequencing,
it preserves and quantifies RNA modifications, revealing detailed
stoichiometric modification profiles and changes after enzymatic treatment,
thereby providing a comprehensive and quantitative view of tRNA sequence and
modification information.
Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection
This study reveals that respiratory syncytial virus (RSV)
infection alters tRNA methylation, specifically demethylating
adenine at position 57 of tRNA-GluCTC, which enables its
cleavage into tRNA-derived fragments (tRFs).
The enzyme AlkB homolog 1 mediates this demethylation,
leading to tRF production that influences RSV replication.
These findings highlight the role of tRNA post-transcriptional
modifications in tRF biogenesis and suggest a novel antiviral
target pathway in RSV infection.
A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides
This work introduces 2D-HELS MS Seq, a two-dimensional
hydrophobic end-labeling strategy that enhances mass
spectrometry-based RNA sequencing by overcoming key technical
challenges in de novo sequencing of mixed and modified RNAs.
The method enables direct sequencing of both canonical and
modified nucleotides, including distinguishing isomeric
modifications such as pseudouridine (Ψ) and uridine (U), and
allows comprehensive analysis of single and mixed RNA samples
without reliance on enzymes or cDNA synthesis.
Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications
This study presents 2D-HELS-AA MS Seq, a direct mass spectrometry-based
RNA sequencing method that eliminates the need for cDNA synthesis and
enables complete sequencing of tRNAPhe while identifying and quantifying
eleven RNA modifications. The method uncovers partial modification
stoichiometries, detects novel truncated isoforms at the 3'-CCA tail,
and discovers a new isoform with A-G base transitions in the variable loop.
By revealing isoforms, base editing, and modification stoichiometries invisible
to traditional sequencing, it offers a powerful new tool for epitranscriptomic
analysis and understanding RNA regulation.
2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
This study demonstrates the 2D-HELS MS Seq method,
which integrates mass-retention time hydrophobic end-labeling
into mass spectrometry for direct, de novo RNA sequencing without
a cDNA intermediate. The technique accurately sequences single
and mixed RNA samples containing both canonical and modified
nucleotides, distinguishing modifications such as pseudouridine (Ψ)
and 5-methylcytosine (m5C) by their unique mass signatures.
A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures
This work introduces 2D-HELS MS Seq, the first direct,
modification-independent RNA sequencing method that combines
hydrophobic end-labeling with mass spectrometry to enable de novo
sequencing of single and mixed RNA samples. The technique
accurately identifies, locates, and quantifies both canonical
and modified bases at single-nucleotide resolution, including
partial modification stoichiometries found in real biological
samples. By overcoming the limitations of cDNA-based sequencing,
it represents a major advance toward comprehensive sequencing of
complex cellular RNAs and provides a valuable tool for quality
control of therapeutic RNAs.
Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms
This study introduces a liquid chromatography-mass spectrometry
(LC-MS) approach that simplifies the de novo sequencing of RNAs
containing noncanonical nucleotides by exploiting consistent
chromatographic separation patterns. Using a two-dimensional
analysis of compound chromatograms and a Monte Carlo algorithm
that generates bidirectional paired-end reads, the method precisely
locates modified nucleotides within RNA sequences. This innovation
overcomes the complexity of conventional MS data caused by multiple
fragmentation pathways and adducts, enabling routine LC-MS sequencing of
both natural and artificially modified RNAs.