BME-seminar 4 - Sequencing the Unseen: Bio-orthogonal 3D Photolitographic Chemistries for Real-Time Molecular Mapping

Abstract: Understanding dynamic molecular interactions in space and time requires tools capable of high-resolution sequencing and multiplexed biomolecule labeling within native cellular environments. Achieving real-time, high-resolution molecular readouts inside living cells remains a central challenge in biology. Here, we present a suite of bio-orthogonal chemistries that integrate live-cell-compatible fluorogenic probes with multiphoton-enabled 3D photolithography for sequencing and spatial tagging in both living and fixed cells. Our system achieves single-nucleotide fidelity through nucleic acid-templated ligation and primer extension reactions that activate fluorescence exclusively upon correct probe–target binding. Using fluorogenic proximity probes for dual-substrate recognition, we extend the platform beyond RNA to detect specific protein complexes. Concurrently, multiphoton-triggered photodeprotection enables submicron spatial resolution for in situ cDNA barcoding and molecular indexing across three dimensions. In living cells, this approach supports real-time tracking of individual RNA transcripts at subsecond intervals; in fixed cells, it enables high-density molecular tagging for downstream sequencing. Together, these innovations offer a transformative framework for decoding the spatiotemporal complexity of gene expression and biomolecular interactions at single-molecule resolution.

Bio: Daniel Fürth is a SciLifeLab Fellow and Assistant Professor at Uppsala University, where he leads a research program focused on bio-orthogonal chemistries and 3D photolithographic technologies for real-time, single-molecule sequencing in living cells. He earned his Ph.D. from Karolinska Institutet and completed his postdoctoral training at Cold Spring Harbor Laboratory in New York. His work bridges synthetic chemistry, advanced microscopy, and spatial genomics to map RNA and protein interactions with high temporal and spatial resolution.