Key Publications

Guo LT et al. Characterization and implementation of the MarathonRT template-switching reaction to expand the capabilities of RNA-seq. RNA (2024). DOI | PubMed

Maio G et al. Improved long-transcript representation in Oxford Nanopore direct RNA sequencing with UltraMarathonRT. bioRxiv (2025). DOI | PubMed

Finding: Demonstrates that substituting UltraMarathonRT into the ONT SQK-RNA004 direct RNA sequencing workflow significantly improves long-transcript representation, yielding higher mean cDNA lengths and greater coverage of full-length molecules compared to the standard ONT reverse transcriptase — establishing uMRT as a performance-enhancing drop-in replacement for nanopore dRNA-seq.

Wiep van der Toorn W et al. Demultiplexing and barcode-specific adaptive sampling for nanopore direct RNA sequencing. Nat. Commun. (2025). DOI | PubMed

Finding: Introduces WarpDemuX, an ultra-fast adapter-barcoding and demultiplexing approach for ONT direct RNA sequencing compatible with SQK-RNA002 and SQK-RNA004 chemistries, demonstrated through multiplexed SARS-CoV-2 transcriptome profiling to identify differences in transcript abundance and poly(A) tail lengths — showcasing the power of multiplexed dRNA-seq for viral RNA biology.

Zhao C, Liu F, Pyle AM. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. RNA (2018). DOI | PubMed

Finding: Foundational characterization of MarathonRT showing it efficiently copies transcripts at least 10 kb in length with superior intrinsic processivity and high fidelity compared to commercial enzymes including SuperScript IV. Identifies the structural basis for this exceptional performance — a loop in the finger subdomain (the α-loop) that acts as a steric guard — and demonstrates that reengineering the enzyme's secondary RNA binding site further enhances its capabilities.

Guo LT et al. Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase. J. Mol. Biol. (2020). DOI | PubMed

Finding: Systematically characterizes and optimizes MarathonRT across multiple applications: single-cycle reverse transcription of long RNAs, in-cell SHAPE-MaP with ultra-long amplicons, DMS-MaP structure probing, and detection of natural RNA base modifications. Demonstrates that MarathonRT generates cDNA templates more than 2.5 kb in length from structure-modified templates where MMLV-based RTs fail after ~700 nt.

Guo LT et al. Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules. Nucleic Acids Res. (2022). DOI | PubMed

Finding: Develops a single-molecule approach to directly track the speed and pausing behavior of MarathonRT and UltraMarathonRT on complex templates in real time, revealing how RT processivity determines the fidelity and completeness of long-RNA sequencing and informing best practices for maximizing full-length transcript recovery.

Ju X et al. Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome. Nature (2024). DOI | PubMed

Finding: Uses MarathonRT-based sequencing to simultaneously determine both termini of individual RNA molecules in Mycobacterium tuberculosis cells, unexpectedly revealing that most Mtb transcripts are incomplete — with 5′ ends at transcription start sites and 3′ ends 200–500 nt downstream due to RNAP pausing. This discovery of a novel transcriptional checkpoint, with implications for TB therapeutics, was enabled by the ability of processive RT sequencing to capture the full spectrum of transcript lengths including short paused species.

Guo LT et al. Sequencing and Structure Probing of Long RNAs Using MarathonRT: A Next-Generation Reverse Transcriptase. J. Mol. Biol. (2020). DOI | PubMed

Finding: First demonstration that MarathonRT enables SHAPE-MaP and DMS-MaP structure probing on long RNAs using ultra-long amplicons, overcoming the fundamental limitation of MMLV-based RTs which can copy only ~700 nt from a SHAPE-modified template — making full structural analysis of large RNAs like lncRNAs and viral genomes tractable for the first time.

Hiers NM et al. An endogenous cluster of target-directed microRNA degradation sites induces decay of distinct microRNA families. Cell Reports (2025). DOI | PubMed

Finding: Identifies a cluster of target-directed microRNA degradation (TDMD) triggers within the Drosophila Kah transcript using Ago1-CLASH, and performs in-cell RNA structure probing of the endogenous trigger cluster using MarathonRT. Structure probing reveals that seed-binding regions reside in accessible single-stranded regions while 3′ end binding regions are more structured — providing mechanistic insight into how TDMD triggers function and demonstrating that MarathonRT enables structure probing of biologically complex loci in living cells.

Sexton AN et al. Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA. Biochemistry (2017). DOI | PubMed

Finding: Foundational study demonstrating that RT termination events (stops) and RT mutation events report on largely non-overlapping sets of DMS-modified nucleotides in chemical probing experiments — showing that combining both signals gives greater structural coverage than either measure alone, and establishing the interpretive framework that MarathonRT-based DMS-MaP protocols build upon.

Huston NC et al. Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms. Mol. Cell (2021). DOI | PubMed

Finding: Applies a novel long-amplicon SHAPE-MaP strategy using MarathonRT to determine the secondary structure of the full 30 kb SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells — the first comprehensive in vivo structural map of a coronavirus genome. Reveals networks of well-folded RNA structures conserved across β-coronaviruses and identifies novel regulatory motifs, including functional stem-loops and the programmed frameshifting pseudoknot, directly from living cell data.

Bohn P et al. Nano-DMS-MaP allows isoform-specific RNA structure determination. Nat. Methods (2023). DOI | PubMed

Finding: Introduces Nano-DMS-MaP, combining DMS mutational profiling with Oxford Nanopore long-read sequencing to resolve structural differences among individual RNA isoforms — a capability impossible with short-read approaches. Applied to HIV-1 transcripts in cells, the method reveals distinct structural landscapes for individual isoforms, demonstrating that MRT's processivity is essential for reading DMS mutations and full-length isoform sequence in a single long sequencing read.

Mitchell D et al. Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS. Nucleic Acids Res. (2023). DOI | PubMed

Finding: Demonstrates that filtering RT mutation signatures by type — distinguishing DMS-induced mismatches from background RT errors — enables high-fidelity RNA structure probing at all four nucleobases using DMS alone, including A, C, G, and U positions. This mutation signature filtering approach is only applicable to MarathonRT and uMRT-based DMS-MaP workflows, extending the structural information content of a single chemical probing experiment.

Saleem et al. Multi-site DMS probing reveals higher-order structure of RNA-protein complexes in living cells. Mol. Cell (2026). DOI | PubMed

Finding: Introduces msDMS-MaP, a single-experiment strategy that simultaneously maps RNA secondary structure, tertiary structure, and protein binding sites in living cells by decoding DMS N7-methylguanine modifications via an optimized reverse transcription protocol that generates a tautomer-induced mutational signature. Applied to the 7SK non-coding snRNP, the method reveals protein binding sites and cooperatively folding tertiary domains — demonstrating that next-generation RT enzymes can decode previously invisible DMS modifications and access a new dimension of RNA structural information.

Tavares RCA et al. MRT-ModSeq - Rapid Detection of RNA Modifications with MarathonRT. J. Mol. Biol. (2023). DOI | PubMed

Finding: Introduces MRT-ModSeq, a method exploiting MarathonRT's sensitivity to RNA modifications under distinct divalent cofactor conditions to generate 2D mutational profiles for rapid, simultaneous detection of multiple modification types in a single sequencing experiment. Demonstrates detection of m1acp3Y, m1A, m3U, m7G, and 2′-OMe modifications in rRNA, and extends to sparse modification sites in MALAT1 lncRNA and PRUNE1 mRNA — establishing MarathonRT as a uniquely capable enzyme for long-read RNA modification sequencing.

Fang et al. A user guide to RT-based mapping of RNA modifications. Methods in Enzymology (2024). DOI | PubMed

Finding: Comprehensive user guide to RT-based approaches for mapping RNA modifications, covering experimental design, choice of reverse transcriptase, cofactor conditions, library preparation, and data analysis. Includes detailed protocols for MarathonRT-based modification mapping methods including MRT-ModSeq, providing researchers with a practical reference for implementing these workflows.

Yamagami et al. Genome-wide analysis of the in vivo tRNA structurome reveals RNA structural and modification dynamics under heat stress. PNAS (2022). DOI | PubMed

Finding: Introduces tRNA structure-seq, a genome-wide workflow using MarathonRT-based mutational profiling to determine in vivo secondary and tertiary structures of tRNAs — a class of RNA that is heavily modified and strongly structured, making it inaccessible to low-processivity RTs. Reveals that tRNA secondary and tertiary structures melt under heat stress and that modification levels change dynamically, establishing tRNA structure-seq as a new tool for studying how tRNA structural and modification landscapes respond to cellular stress.

Chou CL et al. Single-cell RNA-seq using UltraMarathonRT expands the known transcriptome. bioRxiv (2025). DOI | PubMed

Finding: First demonstration of a group II intron reverse transcriptase, UltraMarathonRT, in single-cell RNA-seq, replacing the MMLV-derived RTs used in all current scRNA-seq protocols. Achieves picogram (pg) sensitivity and reveals an unexpected transcriptomics landscape: uMRT-based scRNA-seq captures additional genes and genomic features that conventional RTs miss entirely, expanding the known single-cell transcriptome. Also combines uMRT with metabolic RNA labeling and nucleoside conversion for genome-wide transcriptome dynamics at the single-cell level.

Warkentin R et al. Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT. Bio Protoc (2024). DOI | PubMed

Finding: Describes an improved RT-PCR protocol for circRNA detection using UltraMarathonRT that overcomes two key limitations of existing methods: the inability to reverse-transcribe large, highly structured circRNAs, and the requirement for cloning when sequencing small circRNAs (<150 nt). Unlike MMLV-based RTs, uMRT reverse-transcribes structured circRNAs of any size at ambient temperature, generating concatemeric amplicons that can be sequenced directly by Sanger or Nanopore methods — enabling accurate circRNA validation irrespective of structure, sequence complexity, or length.