Single-cell direct RNA sequencing using electrical zero-mode waveguides and engineered reverse transcriptases - Progress in genome technologies over the past few decades has delivered a dramatic cost reduction in DNA
sequencing and vast increases in read lengths, the latter afforded by development of new single-molecule
sequencing technologies. These advances enabled probing regions of the genome that were considered as
“dark matter” up until recently, as well as the assembly of new high-quality reference genomes. In addition to
genome sequencing, these single-molecule methods have opened up new applications for probing chemical
modifications in DNA, by either probing the kinetics of sequencing-by-synthesis using optical waveguides, or
by electrically distinguishing modified bases using nanopores. Currently, efforts are made to create robust
methods for direct RNA sequencing, so that information about RNA sequence, epigenetic modifications, and
quantity, can be obtained. In a single human cell, only a few picograms of RNA and DNA are available, and
since epigenetic modifications in these nucleic acids cannot be multiplied, a recognized goal of future
sequencing technologies is to reduce the amount of genomic material that can be analyzed at picogram levels.
We have recently developed a method for loading picogram-level DNA and RNA into zero-mode waveguides
(ZMWs), and have demonstrated DNA sequencing of a long DNA fragment, achieved by fabricating porous
ZMWs (PZMWs) in which a porous material was embedded at the ZMW bottoms. However, challenges with
the chemistry and longevity of porous materials have limited the throughput of this system. In this proposal, we
will develop an entirely new method for direct RNA sequencing that enables quantitative transcriptome analysis
and RNA base modification information, requiring only picogram-level input RNA. First, we have developed a
new type of ZMW that contains a metal-disk electrode embedded underneath it. Applying voltage across the
ZMWs produces an electric field that assists with DNA and RNA capture. These new devices allow vastly
increased throughput over the previous generation PZMWs, as well as substantial quality improvements to the
data obtained. Second, for the sequencing engine we will employ MarathonRT, an ultra-processive reverse
transcriptase that converts RNA molecules to complementary DNA (cDNA) molecules by enzymatic replication
robustly and accurately, more so than currently used enzymes used for RNA sequencing. Third, we will employ
advanced single-cell RNA extraction and gold-standard RNA quantification methods. Backed by extensive
preliminary data, we will integrate MarathonRT as the engine, PtZMWs as the sensitive sequence readers and
advanced single-cell sorting and RNA extraction tools, to develop for the first time quantitative RNA expression
profiles from truly single-cell material (i.e., no amplification). Additionally, using our ability to follow the
replication kinetics by MarathonRT, we will probe chemical modifications preserved in these RNA molecules,
such as methyladenine and pseudouridine. Success in this unique approach will revolutionize transcriptome
analysis from single-cell material by providing a workflow for epi/transcriptomics at unprecedented sensitivity.
