Signal transduction with hybridization chain reactions

Some of the most exciting developments in the field of nucleic acid engineering include the utilization of synthetic nucleic acid molecular devices as gene regulators, as disease marker detectors, and most recently, as therapeutic agents. The common thread between these technologies is their reliance on the detection of specific nucleic acid input markers to generate some desirable output, such as a change in the copy number of an mRNA (for gene regulation), a change in the emitted light intensity (for some diagnostics), and a change in cell state within an organism (for therapeutics). The research presented in this thesis likewise focuses on engineering molecular tools that detect specific nucleic acid inputs, and respond with useful outputs.

Four contributions to the field of nucleic acid engineering are presented: (1) the construction of a single nucleotide polymorphism (SNP) detector based on the mechanism of hybridization chain reaction (HCR); (2) the utilization of a single-stranded oligonucleotide molecular Scavenger as a means of enhancing HCR selectivity; (3) the implementation of Quenched HCR, a technique that facilitates transduction of a nucleic acid chemical input into an optical (light) output, and (4) the engineering of conditional probes that function as sequence transducers, receiving target signal as input and providing a sequence of choice as output. These programmable molecular systems are conceptually well-suited for performing wash-free, highly selective rapid genotyping and expression profiling in vitro, in situ, and potentially in living cells.

Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.