3D DNA barcodes for the subcellular profiling of protein expression and distribution

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DNA is a very interesting biomolecule. Our understanding and control of DNA, its structures, properties, and related biological mechanisms have come a long way since the double helix was first described almost 70 years ago. DNA as a linear polymer has a large capacity to store and pass information through its combination of bases. Expanding technologies, including custom synthesis of DNA, PCR to exponentially amplify even a single DNA strand, and next-generation DNA sequencing, have enabled a large amount of information to be engineered into DNA and read out in a highly sensitive and multiplexed manner.

In addition to this primary, linear structure that encodes information, DNA’s complementary hybridization enables its programmable folding into higher-order organizations. This has given rise to the field of DNA nanotechnology, where DNA is used as a material for nanofabrication; 2D and 3D DNA nanostructures of diverse shapes and sizes can be made with exceptional precision.

We feel that these two properties of DNA—its large capacity for information storage and its programmable topology—can complement each other to innovate new applications. In this paper, we harness this multidimensional properties of DNA to develop a sensitive 3D barcoding technology, called sequence-topology assembly for multiplexed profiling (STAMP), for the multiplexed profiling of subcellular protein expression and distribution in cells.

While tools for massively parallel and highly sensitive DNA measurements are widely available, this is not the case for proteins, especially at a subcellular resolution. We believe that this is an important area to apply the two properties of DNA nanostructures in an integrated manner. Specifically, STAMP is a 3D barcoding technology. It utilizes combinatorial sequence content of DNA nanostructures for diverse protein identification and their programmed structural configuration to significantly improve the reaction kinetics of enzymatic and chemical barcoding.  

Leveraging this dual sequence-structure synergy, we couple the DNA nanostructures with short localization labels, exogenous sequences which have differential distributions across subcellular compartments, to form multiplexed STAMP barcodes directly in whole cells. The generated DNA barcodes show improved sensitivity (>100-fold signal enhancement), and can reflect en masse protein expression and subcellular distribution in a high-throughput analysis. When implemented on a miniaturized microfluidic device for clinical applications, STAMP enabled multiplexed protein typing and subcellular distribution analysis in scant patient samples. The STAMP-revealed signatures not only accurately classified cancer molecular subtypes, but also provided new measurements of disease aggressiveness.

Moving forward, we are excited to expand the DNA nanotechnology to other biomedical applications. Such use of DNA nanostructures as a configurable barcoding material not only confers massive information storage, but also has the potential to enable new clinical applications. We anticipate that STAMP could be expanded to detect other biological targets, beyond proteins, with different subcellular distribution to discover new diagnostic and prognostic biomarker signatures, thereby improving patient stratification and treatment decision.

Written by Noah Sundah and Huilin Shao

Our paper:
Sundah et al. Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution. Nature Biomedical Engineering (2019)

News & views:
Zhang et al. Barcodes for subcellular protein localization. Nature Biomedical Engineering (2019)

Huilin Shao

Assistant Professor, National University of Singapore