Nanopore microscope identifies RNA isoforms with structural colours

Abstract

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers (IDs) with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colors. Structural colors consist of DNA structures, protein labels, native RNA structures, or a combination of all three. The sequence of structural colors of RNA IDs enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) reveals structural arrangements in native transcripts in agreement with published variants.ARTEMIS discriminates circular and linear transcript isoforms in a one step, enzyme-free reaction in a complex human transcriptome using single-molecule readout. Main TextSingle-molecule identification of multiple transcript isoforms in parallel without preamplification is critical for understanding transcriptome diversity and gene expression networks 1 . Identification and quantification of structural arrangements in native transcripts are both challenging, and current methods do not necessarily yield results reflecting innate transcriptome diversity 2,3 . Although identification of long RNA molecules is possible with existing nucleic acid detection methods 4-6 , they lack specificity and simplicity. In addition, common approaches mainly rely on enzymatic reactions and require preamplification. These lead to inevitable biases and loss of information [7][8][9] . RNA sequencing approaches require extensive and intricate adaptations to achieve the sequencing of transcript variants and to test their circularity 2,10,11 . These widely used techniques face amplification and reverse transcription biases, and detection of transcript variants is affected by short reads in RNA sequencing 10,11 .Recently, nanopore sequencing introduced direct RNA readout 10 , however, access to singlemolecule information of gene expression level in combination with low-quality reads and uncertainty about the 5'end of the transcript remain major challenges 12 . As in previously established RNA-seq methods, nanopore RNA-seq also suffers from enzymatic biases 13,14 .Additionally, secondary structures in both RNA and complementary DNA (cDNA) contribute to biases by obstructing the binding of primers and sequencing adapters 15 . In a vast majority of cases for identifying disease-specific transcripts looking for a transcriptome subset is sufficient 39

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

“…Solid-state nanopore based experiments are easy to prepare and quick to perform, and data analysis is less time-consuming; they have been used previously to detect RNA conformations. 78 − 84 However, all these studies were performed at salt concentrations (commonly between 0.3 and 1 M KCl) significantly higher than physiologically relevant levels, and hence it is likely that the conformations of the RNAs will differ from those formed under physiological ionic strength conditions. 60 Our polymer–electrolyte nanopore system uncouples the solution used to dilute the analyte from the bath solution and it provides an opportunity to study RNAs in electrolyte which facilitates the refolding of RNA into native structures under physiologically relevant conditions.…”

Emerging Approaches to DNA Data Storage: Challenges and Prospects

“…The correct identification of the “color” was verified using fluorescence microscopy, wherein fluorescently labeled (5′-fluorescein) structural units were used. (C,D) Images reproduced with permission under a Creative Commons Attribution 4.0 License (CC BY) from ref ( 130 ). Copyright 2022 Springer Nature.…”

“… 102 Increased storage density beyond binary barcodes can also be achieved by creating blocks of repeating structural units that appear as a single protrusion within the nanopore, which creates “structural colors” to generate up to 10 data levels. 130 Compared with fluorescence, sequencing, or gel electrophoresis-based strategies, single-molecule nanopore measurements require less material and enable faster data reading; through a combination of this technology with deep learning methods, 131 real-time nanopore data analysis is attainable.…”

Nanopore Deciphering Single Digital Polymers Towards High‐Density Data Storage

“…[29] Copyright (2021), with permission from Wiley‐VCH. (d) Identification of RNA targets using multiple structural colors of DNA/RNA complexes [30] . Left: the schematic structure of DNA/RNA complexes labelled with distinct structural colors.…”

“…[27] More recently, the variable numbers of the nanoscale structural units per zone were used to define different bits to determine the identifiers (IDs) of RNA isomers (Figure 3d). [30] For instance, a nanostructure zone owning 1, 2 or 3 structural units represent the bit-1, bit-2 or bit-3, which create the structural color "1", "2" or "3", respectively. For each structural unit, the first 20 bases of a docking DNA strand are complementary to the specific sequence of the target RNA, and the other 20 bases are complementary to an imaging DNA strand that bound to a monovalent streptavidin via its biotinylated 3'end.…”