In Situ Signal Amplification for Spatial Transcriptomics Using Programmable DNA Assemblies

Citation

Colón, Katsuya Lex (2025) In Situ Signal Amplification for Spatial Transcriptomics Using Programmable DNA Assemblies. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/pp5f-pk64. https://resolver.caltech.edu/CaltechThesis:03312025-203601435

Abstract

Sequential Fluorescent In Situ Hybridization (seqFISH) has been an invaluable tool in imaging-based spatial transcriptomics, aiding researchers in elucidating spatially-resolved, gene expression patterns in intact tissues and cell culture models. However, methods that rely on smFISH, such as seqFISH, suffer from poor signal-to-noise ratio in certain tissue types or target RNA, require many fluorescently labeled RNA targeting probes which prohibits imaging of small RNA species, and exhibit poor sample throughput due to the need of high magnification objective or long exposure times. Herein, we develop solutions to these limitations by developing and utilizing a robust signal amplification strategy. While various amplification technologies exist, their limitations often hinder broad applicability. Moreover, we desire an amplification platform that is amenable to the denaturing wash conditions used in seqFISH. We will begin Chapter I by discussing the background, technical challenges, and utility of various in situ signal amplification technologies. Chapter II details the exploration and technical limitations of rolling circle amplification (RCA) and branched DNA (bDNA) assembly utilizing ssDNA padlock amplifier strands. Chapter III discusses the design and development of a novel amplification strategy called Signal amPlicAtion by Recursive Crosslinking (SPARC), which builds upon the knowledge gained from Chapter II. We highlight SPARC as a unique photochemical signal amplification method that iteratively deposits amplifier strands near the primary probe target for linear signal amplification. Then, the deposited amplifier strands act as a scaffold for branched DNA assembly, leading to an exponential signal amplification. Through each deposition and assembly step, amplifier strands are photo-crosslinked to the extracellular matrix, forming highly stable DNA nanostructures that can withstand harsh denaturing wash conditions. We demonstrate the utility of SPARC in amplifying signal of both single-molecule transcripts and proteins.

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