Projects

Biological nanopores have been used as powerful platforms for label-free detection and identification of a range of biomolecules for biosensing applications and single-molecule biophysics studies. Nonetheless, high limit of detection (LOD) of analytes due to inefficient biomolecular capture into biological nanopores at low voltage poses practical limits on their biosensing efficacy. Traditionally, biological nanopores are incorporated into large planar lipid membranes painted on polymer apertures (PTFE, Delrin). Although convenient for research, these membranes are typically sensitive to vibrational noise, not stable over time, and further, do not survive at a high voltage bias. Here, we developed a chip-based platform that allows the formation of long-lived lipid bilayer membranes of comparable areas to traditional systems, even under high sustained voltages of 350 mV. Using this platform, we demonstrate sensing of DNA hairpins in 𝛼-hemolysin nanopores at the nanomolar regime under high voltage. Further, we have developed a workflow for one-pot enzymatic release of DNA hairpins with different stem lengths from magnetic microbeads, followed by multiplexed nanopore-based quantification of the hairpins within minutes, paving the way for novel nanopore-based multiplexed biosensing applications.

In recent years, nanopore-based sequencers have become robust tools with unique advantages for genomics applications. However, progress towards applying nanopores as highly sensitive, quantitative diagnostic tools has been impeded by several challenges. One major limitation is the insufficient sensitivity of nanopores in detecting disease biomarkers, which are typically present at pM or lower concentrations in biological fluids, while a second limitation is the general absence of unique nanopore signals for different analytes. To bridge this gap, we have developed a strategy for nanopore-based biomarker detection that utilizes immunocapture, isothermal rolling circle amplification, and sequence-specific fragmentation of the product to release multiple DNA reporter molecules for nanopore detection. These DNA fragment reporters produce sets of nanopore signals that form distinctive fingerprints, or clusters. This fingerprint signature therefore allows the identification and quantification of biomarker analytes. As a proof of concept, we quantify human epididymis protein 4 (HE4) at low pM levels in a few hours. Future improvement of this method by integration with a nanopore array and microfluidics-based chemistry can further reduce the limit of detection, allow multiplexed biomarker detection, and further reduce the footprint and cost of existing laboratory and point-of-care devices

Pseudouridine (psi) is one of the most highly abundant RNA modifications. While extensive studies have been carried out on transfer RNA, ribosomal RNA, and small nucleolar RNA, the physiological effect of psi on mRNAs has remained unclear. Recent research shows a relationship between environmental signals such as nutrient deprivation with psi modification. Evidence also suggests a role of psi modifications on splicing regulation, regulation of mRNA stability, and regulation of translation. In mammalian cells, TRUB1 is the predominant psi synthase acting on mRNAs. Our direct RNA nanopore sequencing studies have unveiled several hypermodified mRNA for which >40% of transcripts are psi-modified at a given site by TRUB1 (AK2, CFC6, GTF3, IDI1, MCM5 and PFKP). To gain deeper insight into the biological function of psi modifications, we assess the subcellular localization of these mRNAs that are highly modified by TRUB1 using single-molecule RNA fluorescence in situ hybridization (smFISH) on human cells. Furthermore, we study the effects of TRUB1 knockdown on the subcellular localization and expression levels of these mRNA with single-cell resolution. Finally, we examine the rate of mRNA decay as a function of TRUB1 expression. Our results provide valuable information about the biological significance of the ubiquitous psi modification in mRNA.