Biology subjects

Nian, K.

Publications and source records attributed to Nian, K..

4 recordsLinked to original sources

bioRxiv2024

NuclampFISH enables cell sorting based on nuclear RNA expression for chromatin analysis

Transcriptional bursts refer to periods when RNA polymerase interacts with a DNA locus, leading to active gene transcription. This bursting activity can vary across individual cells, and analyzing the differences in transcription sites can help identify key drivers of gene expression for a specific target. Scaffolding methods based on fluorescence in situ hybridization (FISH) have been widely used to amplify the fluorescent signal of mRNAs and sort cells based on mRNA expression levels. However, these methods are inefficient at targeting nuclear RNA, including transcription sites, due to the probes limited accessibility through cellular compartment membranes and crosslinked proteins. Additionally, the required formaldehyde fixation interferes with downstream analysis of chromatin and protein-binding interactions. To address these challenges, a platform that integrates amplified FISH with reversible crosslinkers and allows access to the nucleus is needed. In response, we developed nuclear clampFISH (nuclampFISH). This method amplifies the fluorescent signal of mRNAs using a reversible crosslinker, enabling the sorting of cells based on nuclear RNA expression and compatible with downstream biochemical analysis. This assay demonstrates that transcriptionally active cells have more accessible chromatin for a respective gene. Notably, the tools developed are highly accessible and do not require specialized computation or equipment.

genomics↗

bioRxiv2024

Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity

Chemical modifications in mRNAs, such as pseudouridine (psi), can control gene expression. Yet, we know little about how they are regulated, especially in neurons. We applied nanopore direct RNA sequencing to investigate psi dynamics in SH-SY5Y cells in response to two perturbations that model a natural and unnatural cellular state: retinoic-acid-mediated differentiation (healthy) and exposure to the neurotoxicant, lead (unhealthy). We discovered that the expression of some psi writers change significantly in response to physiological conditions. We also found that globally, lead-treated cells have more psi sites but lower relative occupancy than untreated cells and differentiated cells. Interestingly, examples of highly plastic sites were accompanied by constant expression for psi writers, suggesting trans-regulation. Many positions were static throughout all three cellular states, suggestive of a "housekeeping" function. This study enables investigations into mechanisms that control psi modifications in neurons and its possible protective effects in response to cellular stress.

systems biology↗

bioRxiv2022

Paired aptamer capture and FISH detection of individual virions enables cell-free determination of infectious titer

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titer is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and reverse transcription polymerase chain reaction (RT-PCR)-based (sensitive but not rapid). Current viral titer methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid-aptamer FISH or raptamer FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titer. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer, then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH)- thus, it is selective for infectious particles (i.e., positive for coat protein and positive for genome).

molecular biology↗

bioRxiv2022

DNA-enhanced CuAAC ligand enables live-cell detection of intracellular biomolecules

Of the various conjugation strategies for cellular biomolecules, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the preferred click chemistry approach due to its fast reaction rate and the commercial availability of a wide range of conjugates. While extracellular labeling of biomolecules using CuAAC has been widely adopted, intracellular labeling in live cells has been challenging as the high copper concentrations required for CuAAC reaction is toxic to biological systems. As a critical first step towards CuAAC-mediated intracellular labeling, an ultrasensitive CuAAC ligand is needed to reduce cytosolic copper concentrations while maintaining fast reaction kinetics. Here, we developed BTT-DNA, a new DNA oligomer-conjugated CuAAC ligand for click reaction biomolecular labeling. The DNA oligo attachment serves several purposes, including: 1. Increased localization of copper atoms near the ligand, which enables ligation of azide tags with much lower copper concentrations than commercially available CuAAC ligands and without the addition of exogenous copper salt; 2. Allows nucleic acid template-driven proximity ligation by choosing the attached DNA sequence, 3. Enables the liposome encapsulation and delivery of the ligand into live cells, and 4. Facilitates intracellular labeling of nascent phospholipids in live cells. We demonstrate that BTT-DNA mediated labeling has little to no effect on the overall cell health.

molecular biology↗