Single-Molecule High-Confidence Detection of miRNA Cancer Biomarkers
PROJECT SUMMARY/ ABSTRACT
The ultimate goal of this proposal is to develop a technology platform for high confidence and robust single
molecule analysis of miRNA biomarkers in cancer samples through simultaneous detection of miRNAs, in
under an hour. Although miRNAs are short, they regulate essentially all cellular pathways relevant to human
health and disease, including cancer. After being exported from cells, the cell-free circulating miRNAs are
found to be relatively more stable than other nucleic acids, making them of high interest as clinical cancer
biomarkers. Current methods for miRNA analysis, including PCR assays, face challenges due to inherent
inconsistencies in day-to-day and lab-to-lab results in addition to false negatives and positives. We have
recently developed a unique fluorescence resonance energy transfer (FRET)-based single molecule dynamic
sensor to enable high confidence and ultrasensitive detection of an unlabeled DNA as well as miRNA targets
and demonstrated that the sensing platform works in serum and fully discriminates targets from point mutant
controls. Using total internal reflection fluorescence microscopy, we demonstrated that the sensor exhibits a
static FRET level in the absence of a target. However, in the presence of the target, the sensor forms a four-
way junction and hence undergoes a dynamic switching between a low- and a high-FRET state, a feature
that enables high-confidence detection of the target. We demonstrated these features initially using a p53
tumor suppressor gene and later a miRNA associated with the triple-negative breast cancer (TNBC), both in
buffer and in spiked-in samples using 10% serum. In this proposal, we will focus on the development and
testing of this platform for high-confidence detection through multiplexed analysis of miRNAs in non-clinical
as well as minimally processed cancer samples. In Aim 1, we will design and characterize a sensing platform
for the detection of DNA sequences in one sample. In Aim 2, we will characterize and validate the detection
platform for simultaneous detection of miRNAs specific to TNBC. We will also establish a speedy detection
of TNBC miRNAs using a microfluidic device with parallel channels. In Aim 3, we will identify the abundant
miRNAs in tumor tissues and serum samples of TNBC-carrying patient-derived xenograft (PDX) mice via
miRNA sequencing and apply our single-molecule multiplexed platform to detect those TNBC miRNAs in
serum samples from the PDX mice. Our proposed approach offers a number of important innovations
including i) a generic platform for error-free detection of miRNAs, ii) ultimate sensitivity via single-molecule
detection, iii) simultaneous detection of multiple biomarkers in the same sample - allowing high-confidence
detection, and iv) target labeling and amplification are not required. Therefore, this multiplexed platform has
the potential to be a transformative technology in the early diagnosis of cancer. By providing detection
sensitivity and confidence that meets or exceeds state-of-the-art clinical analyzers, the proposed approach
could bring new initiatives in clinical diagnosis, cancer assessment, and individualized cancer treatments.