Biology subjects

Prescher, J. A.

Publications and source records attributed to Prescher, J. A..

11 recordsLinked to original sources

bioRxiv2026

A miniature bioluminescent platform for imaging structured RNAs

A broader understanding of RNA biology requires methods for visualizing transcript dynamics in native environments. Common imaging approaches rely on fluorescent probes that require excitation light and often depend on large aptamer arrays, limiting sensitivity and compatibility with many non-coding RNAs, particularly highly structured folds. To address these challenges, we developed a miniaturized bioluminescent platform for RNA imaging. This system employs a compact RNA tag that assembles luciferase fragments upon binding of monomeric RNA-binding proteins (RNA lanterns). Systematic optimization of the RNA component yielded a structured 24-nucleotide tag, representing greater than 3-fold reduction in size relative to the original C-11-B RNA tag, placing it among the smallest RNA imaging tags reported to date. The optimized tag enabled RNA-dependent bioluminescence detection in live cells, notably as a small, single insert within highly structured non-coding RNAs, including the MALAT1 ENE domain and a small HDV-like ribozyme. Collectively, this work establishes a miniaturized bioluminescent platform for imaging a broad range of RNA targets in living systems.

molecular biology↗

bioRxiv2025

An open, integrated platform for multiplexed bioluminescence microscopy

We report an imaging package to democratize all-in-one bioluminescence and fluorescence microscopy. The platform comprises three tools: PhasorViewer, a visualization suite to design experiments and identify optimal probe combinations; PhasorScope, an open-source, cost-effective microscopy framework to upgrade conventional microscopes; and PhasorAnalysis, a dedicated, user-friendly analysis pipeline to quantify phasor imaging data. We demonstrate the utility of the platform for multiplexed, simultaneous fluorescence and bioluminescence imaging with readily accessible optical reporters.

bioengineering↗

bioRxiv2025

Continuous hypermutation and evolution of luciferase variants

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, "winning" mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a non-cognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/669707v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3959dborg.highwire.dtl.DTLVardef@13a57c0org.highwire.dtl.DTLVardef@1572c04org.highwire.dtl.DTLVardef@abd59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Bioorthogonal cyclopropenones for investigating RNA structure

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments. Existing tools primarily rely on electrophiles that are constitutively "on" or triggered by UV light, often resulting in high background reactivity. We developed an alternative, chemically triggered approach to crosslink RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes--electrophiles that can trap neighboring nucleophiles to forge covalent crosslinks. As proof-of-concept, we synthesized a panel of CpOs and appended them to thiazole orange (TO-1). The TO-1 conjugates bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, confirmed by fluorescence turn-on. After phosphine administration, covalent crosslinks were formed between the CpO probes and RNA. The degree of crosslinking was both time and dose-dependent. We further applied the chemically triggered tools to model RNAs in biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

biochemistry↗

bioRxiv2024

ESPRESSO: Spatiotemporal omics based on organelle phenotyping

Omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, have been instrumental to improving our understanding of complex biological systems. Despite fast-pace advancements, a crucial dimension is still left to explore: time. To capture this key parameter, we introduce ESPRESSO (Environmental Sensor Phenotyping RElayed by Subcellular Structures and Organelles), a pioneering technique that provides high-dimensional phenotyping resolved in space and time. Through a novel paradigm, ESPRESSO combines fluorescent labeling, advanced microscopy and bioimage and data analysis to extract morphological and functional information of the organelle network unveiling phenotypic changes over time at the single-cell level. In this work, we present ESPRESSOs methodology and its application across numerous cellular systems, showcasing its ability to discern cell types, stress response, differentiation and immune cells polarization. We then correlate ESPRESSO phenotypic changes with gene expression and demonstrate its applicability to 3D cultures, offering a path to revolutionizing biological exploration, providing invaluable insights into cellular states in both space and time.

bioengineering↗

bioRxiv2024

Monitoring macrophage polarization with gene expression reporters and bioluminescence phasor analysis

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function, but remains a challenging pursuit. Existing methods are mostly limited to static readouts or difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2-specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernable in both 2D and 3D formats with single cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.

bioengineering↗

bioRxiv2022

A modular platform for bioluminescent RNA tracking

A complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we developed a bioluminescent platform for serial imaging of RNAs. Small RNA tags were engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production was observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag was necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

molecular biology↗

bioRxiv2021

Longitudinal monitoring of individual infection progression in Drosophila melanogaster

The innate immune system is critical for host survival of infection. Infection models in organisms like Drosophila melanogaster are key for understanding evolution and dynamics of innate immunity. However, current toolsets for fly infection studies are limited in their throughput and their ability to generate longitudinal measurements of infection progression in single animals. Here we report a novel bioluminescent imaging strategy enabling non-invasive characterization of pathogen load over time. We demonstrate that photon flux from autobioluminescent reporter bacteria can be used to monitor relative pathogen loads in individual animals. Escherichia coli expressing the ilux operon were imaged in whole, living flies at relevant concentrations for immune study. Because animal sacrifice was not necessary to estimate pathogen load, stochastic responses to infection were characterized in individuals over time for the first time. The high temporal resolution of bioluminescence imaging also enabled visualization of the fine dynamics of microbial clearance on the hours time-scale. Overall, this non-invasive imaging strategy provides a simple and scalable platform to observe changes in pathogen load in vivo over time.

immunology↗

bioRxiv2021

Multiplexed bioluminescence microscopy via phasor analysis

Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.

bioengineering↗

bioRxiv2021

Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer

Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are often complicated by the underlying heterogeneity of the cell types involved. Here we performed a comprehensive analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. We created a suite of organ-derived metastatic cell lines from the MMTV-PyMT mouse model. Bulk sequencing analyses uncovered tissue-specific genes across the different metastatic and primary tumor samples. We further investigated intratumoral heterogeneity by performing single-cell RNA-seq. These data revealed transcriptomes that may contribute to sub-clonal evolution during cancer progression. Collectively, the organ-derived cell lines provide a platform for comprehensive studies of metastatic breast cancer progression.

cancer biology↗

bioRxiv2019

Rapid multicomponent bioluminescence imaging via substrate unmixing

Engineered luciferases and luciferins have dramatically expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Here we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale, a vast improvement over conventional protocols.

bioengineering↗