Biology subjects

Patino-Guillen, G.

Publications and source records attributed to Patino-Guillen, G..

2 recordsLinked to original sources

bioRxiv2025

Quantification of disease-associated RNA tandem repeats by nanopore sensing

Short tandem repeat expansions underlie a class of neurological and neuromuscular diseases known as repeat expansion disorders (REDs), yet the precise characterization of these repeats remains technically challenging. Conventional amplification-based methods fail to resolve repeat length accurately due to amplification bias and sequence homogeneity. Here, we present a single-molecule nanopore-based strategy that enables direct quantification of tandem repeats in native RNA. By assembling RNA:DNA nanostructures that encode specific repeat number, we achieve repeat size discrimination with a resolution of 18 nucleotides. Using tandem repeat-containing RNA, we successfully detect and discriminate disease-relevant repeat lengths associated with myotonic dystrophy types 1 (DM1) and 2 (DM2), and congenital central hypoventilation syndrome-1 (CCHS1). Finally, we apply our method to total RNA extracted from a DM1 human cell line model, demonstrating its compatibility with complex biological samples. Our approach offers a platform for studying repeat expansion biology at the single-molecule level, with broad implications for diagnostics, clinical research and multiplexed repeat profiling.

biochemistry↗

bioRxiv2023

Single-Molecule RNA Sizing Enables Quantitative Analysis of Alternative Transcription Termination

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilized T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discovered alternative transcription termination in the origin of replication sequence. Next, we employed circular DNA without transcription terminators to perform rolling circle transcription. This allowed us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.

biophysics↗