Biology subjects

Schulte, S. J.

Publications and source records attributed to Schulte, S. J..

5 recordsLinked to original sources

bioRxiv2025

Models and Algorithms for Equilibrium Analysis of Mixed-Material Nucleic Acid Systems

Dynamic programming algorithms within the NUPACK software suite enable analysis of equilibrium base-pairing properties for complex and test tube ensembles containing arbitrary numbers of interacting nucleic acid strands. Currently, calculations are limited to single-material systems that are either all-RNA or all-DNA. Here, to enable analysis of mixed-material systems that are critical for modern applications in vitro, in situ, and in vivo, we develop physical models and dynamic programming algorithms that allow the material of the system to be specified at nucleotide resolution. Free energy parameter sets are constructed for both RNA/DNA and RNA/2'OMe-RNA mixed-material systems by combining available empirical mixed-material parameters with single-material parameter sets to enable treatment of the full complex and test tube ensembles. New dynamic programming recursions account for the material of each nucleotide throughout the recursive process. For a complex with N nucleotides, the mixed-material dynamic programming algorithms maintain the O(N 3) time complexity of the single-material algorithms, enabling efficient calculation of diverse physical quantities over complex and test tube ensembles (e.g., complex partition function, equilibrium complex concentrations, equilibrium base-pairing probabilities, minimum free energy secondary structure(s), and Boltzmann-sampled secondary structures) at a cost increase of roughly 2.0-3.5x . The results of existing single-material algorithms are exactly reproduced when applying the new mixed-material algorithms to single-material systems. Accuracy is significantly enhanced using mixed-material models and algorithms to predict RNA/DNA and RNA/2'OMe-RNA duplex melting temperatures from the experimental literature as well as RNA/DNA melt profiles from new experiments. Mixed-material analyses can be performed online using the NUPACK web app (www.nupack.org) or locally using the NUPACK Python module.{paragraph} O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/662484v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1363811org.highwire.dtl.DTLVardef@9af312org.highwire.dtl.DTLVardef@cc4d1corg.highwire.dtl.DTLVardef@da876c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples

Signal amplification based on the mechanism of hybridization chain reaction (HCR) provides a unified framework for multiplex, quantitative, high-resolution imaging of RNA and protein targets in highly autofluorescent samples. With conventional bandpass imaging, multiplexing is typically limited to four or five targets due to the difficulty in separating signals generated by fluorophores with overlapping spectra. Spectral imaging has offered the conceptual promise of higher levels of multiplexing, but it has been challenging to realize this potential in highly autofluorescent samples including whole-mount vertebrate embryos. Here, we demonstrate robust HCR spectral imaging with linear unmixing, enabling simultaneous imaging of 10 RNA and/or protein targets in whole-mount zebrafish embryos and mouse brain sections. Further, we demonstrate that the amplified and unmixed signal in each of 10 channels is quantitative, enabling accurate and precise relative quantitation of RNA and/or protein targets with subcellular resolution, and RNA absolute quantitation with single-molecule resolution, in the anatomical context of highly autofluorescent samples. SUMMARYSpectral imaging with signal amplification based on the mechanism of hybridization chain reaction enables robust 10-plex, quantitative, high-resolution imaging of RNA and protein targets in whole-mount vertebrate embryos and brain sections.

developmental biology↗

bioRxiv2023

Multiplex, quantitative, high-resolution imaging of protein:protein complexes via hybridization chain reaction

Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with high signal-to-background in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of 3 different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with 1-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/550181v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@dee6f4org.highwire.dtl.DTLVardef@cc6a6org.highwire.dtl.DTLVardef@c5ea79org.highwire.dtl.DTLVardef@1a4edee_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

HCR Lateral Flow Assays for Amplified Instrument-Free At-Home SARS-CoV-2 Testing

The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/L using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/L, 1000 copies/L, 2000 copies/L, 2000 copies/L, and 20,000 copies/L. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious disease. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection. HCR lateral flow assayO_LIAmplified C_LIO_LIInstrument-free C_LIO_LIAt-home C_LIO_LI60 min C_LIO_LINaked eye C_LIO_LISARS-CoV-2 C_LIO_LI200 copies/L C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/508442v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@a04874org.highwire.dtl.DTLVardef@f4ba0borg.highwire.dtl.DTLVardef@e2445corg.highwire.dtl.DTLVardef@f63411_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

RNA in situ hybridization (RNA-ISH) based on the mechanism of hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we extend the benefits of 1-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry (IHC), enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. More-over, we provide a unified framework for simultaneous quantitative protein and RNA imaging with 1-step HCR signal amplification performed for all target proteins and RNAs simultaneously. SUMMARYSignal amplification based on the mechanism of hybridization chain reaction enables multiplexed, quantitative, high-resolution imaging of protein and RNA targets in highly autofluorescent tissues.

developmental biology↗