Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis

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TITLE: Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine TR&D 1: Single-Molecule Processing: Detection and Identification of Single DNAs, RNAs, and Proteins using Immobilized Nanoscale Enzymatic Reactors (INERs) and Nanoscale Electrophoresis Abstract/Summary The ability to process single molecules has already demonstrated its utility in a number of basic and translational endeavors in biology and medicine. There are tangible examples of its success including digital PCR (dPCR) and Next Generation Sequencing (NGS). In the case of dPCR, samples are parsed into nanoliter volumes such that each reactor volume contains statistically a single molecule, which is subsequently amplified via PCR. This technique shows exquisite analytical sensitivity by discerning subtle target copy number variations. For NGS, bridge PCR is used to create clonal clusters of amplified targets for sequencing-by-synthesis. Unfortunately, both do require a PCR step, which can be problematic. For example, amplification can mask epigenetic modifications in DNA and/or RNA that can carry important diagnostic and/or prognostic information for disease management (i.e., Precision Medicine). While amplification-free strategies are preferred, this can be problematic when analyzing clinical markers that are sometimes low in abundance. This is the case when attempting to analyze blood-borne markers, such as the liquid biopsy markers. For example, a single circulating tumor cell (CTC) carries 6 pg of genomic DNA and thus, may not be detected by NGS without significant rounds of amplification. In this P41 competitive renewal application of CBM2, the Center will develop a suite of tools that can process single molecules (DNAs, RNAs, and proteins) harvested from liquid biopsy markers, such as CTCs, and extracellular vesicles (EVs), using amplification-free strategies. The unique attributes of our tools is that they will not only detect, but also identify unamplified single molecules with high efficiency. In TR&D 1, immobilized nanoscale enzymatic reactors (INERs) will be realized that can enzymatically digest DNAs (using Exo I processive exonuclease), RNAs (uses XRN1, a processive exoribonuclease), and proteins (trypsin, which is a proteolytic enzyme). A fluidic network fabricated in a plastic via nanoimprint lithography (NIL) will be generated that contains a sub-micron pillar to which the enzyme is surface immobilized. The INERs can be connected to nanoscale electrophoresis that can monitor in real time the reaction products with high identification accuracy via their electrophoretic mobility (i.e., Time-of-Flight, TOF) using fluorescence single-molecule tracking during their electrokinetic transport through a plastic-based nano-column. Unique phenomena occurring in the nanometer electrophoresis columns will produce molecular-dependent mobilities that are not observed using microscale columns. Coupled with outputs from TR&D 2 (in-plane nanopore sensors), the INER products can potentially be detected using a label-free approach. An application scenario that will be demonstrated using INERs coupled to nanoscale electrophoresis is the ability to identify membrane proteins in liquid biopsy markers, such as extracellular vesicles (EVs). Because we are working with non-amplified targets, the tools generated by the Center will have the ability to directly detect and identify molecular signatures that are hard to read using amplification strategies, such as low abundance proteins.