Tunneling Spectroscopy on Engineered Surfaces for RNA Nucleotide and Structural Label Identification

Although several advances have been made in RNA sequencing and structural characterization, the lack of a method for directly determining the sequence and structure of single RNA molecules has limited our ability to probe heterogeneity in gene expression at the level of single cells. We present a method for identification of RNA nucleotides and structural labels for mapping of single RNA molecules. With non-perturbative tunneling spectroscopy, we probed the molecular orbitals of distinct nucleobases within RNA macromolecules immobilized with restricted conformational freedom on a chemically modified surface. From the measurements, models for tunneling were combined to obtain twelve biophysical parameters unique to the ribonucleotides within the electronic junction [1]. The twelve parameters serve as a comprehensive molecular fingerprint, enabling ribonucleotide discrimination and identification of structure-dependent chemical labels with machine learning. We show high accuracy for both ribonucleotide discrimination (>99%) and chemical label identification (>98%) with a modest 35 repeat measurements [2]. This technique facilitates probing the transcriptome with a previously unattainable level of detail.

[1] Korshoj, et al. Small 2017; [2] Abel, Jr., et al. Chemical Science 2019