DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling

Abstract

Molecular biomarkers play a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. Detecting multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. Here, we report programmable DNA nanoswitches for multiplexed detection of up to 6 biomarkers at once with each combination of biomarkers producing a unique barcode signature among 64 possibilities. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules, for example, DNA, RNA, antibody, and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen.

Keywords: DNA barcodes; DNA nanoswitches; biosensing; diagnostics; genotyping; multiplexed detection.

Figure 1.. DNA nanoswitch barcodes.

Schematic illustration showing construction and use of DNA nanoswitches to yield barcoded results for diagnostic assays.

Figure 2.. DNA nanoswitch overview and characterization.

(a) Reconfiguration of the DNA nanoswitch in the presence of a nucleic acid target turning from a linear “off” state to a looped “on” state. The two states of the nanoswitch can be readout on an agarose gel (inset). (b) Demonstration of detecting different gene fragments using the DNA nanoswitch assay. (c) Sensitivity analysis of DNA nanoswitch assay for the cystic fibrosis (CF) gene fragment. (d) Specificity of DNA nanoswitches against CF gene fragments containing one to three mismatches.

Figure 3.. DNA nanoswitch barcodes for multiplexed detection of nucleic acids.

(a) Design of the DNA nanoswitch with variable regions (V1-V12) that allow programmable placement of detectors, resulting in nanoswitches with different loop sizes. (b) A mixture of nanoswitches with different loop sizes yields a multiplexed gel readout of up to 6 gene fragments (inset). Each target nucleic acid triggers the formation of a specific loop, providing a unique signal (targets are colored for clarity). (c) Demonstration of lack of cross-reactivity in nanoswitches designed for 6 different gene fragments. (d) Full set of DNA nanoswitch barcodes showing detection of all possible combinations of the 6 gene fragments.

Figure 4.. Detecting multiple types of biomarkers using DNA nanoswitch barcodes.

(a) Using small molecules such as biotin and digoxygenin to target macromolecules. (b) Strategy to detect nucleic acids (DNA and RNA), a protein (streptavidin) and an antibody (anti-digoxygenin). (c) A nanoswitch mixture to demonstrate simultaneous detection of four different biomarkers. (d) Demonstration of lack of crosstalk in nanoswitches designed for four different target biomolecules. (e) Full set of DNA nanoswitch barcodes showing detection of all possible combinations of anti-digoxygenin (anti-dig), an RNA sequence, a DNA sequence and streptavidin. (f) Individual targets can be analyzed even in the presence of other target biomolecules, showing a “multiplexed non-interference” in the barcoded assay.

Figure 5.. DNA nanoswitch barcodes for prostate cancer biomarker panel.

(a) An illustration of the three chosen prostate cancer biomarkers (miR-141, miR-30c and PSA). (b) DNA nanoswitch mixture for detecting prostate cancer biomarker panel. (c) Barcode detection of individual biomarkers (DNA analogs of microRNAs 141 and 30c, and PSA) and simultaneous detection of all three biomarkers in buffer and 20% fetal bovine serum (FBS).