Biology subjects

Miao, S.

Publications and source records attributed to Miao, S..

4 recordsLinked to original sources

bioRxiv2022

Fluorogenic U-rich internal loop (FLURIL) tagging with bPNA enables intracellular RNA and DNA tracking

We introduce herein a new strategy for intracellular RNA and DNA tracking that is robust, orthogonal and complementary to existing methods: Fluorogenic U-Rich Internal Loop (FLURIL) tagging with cell-permeable fluorophore-labeled bifacial Peptide Nucleic Acids (fbPNAs). Our approach uses an 8-nt (U4xU4) U-rich internal loop (URIL) in the RNA of interest (ROI) as a compact labeling site for fluorogenic triplex hybridization with a bPNA probe (~1 kD). FLURIL tagging thus replaces a 4 bp duplex stem with a labeled 4-base-triple hybrid stem of similar structure and mass. In contrast to existing strategies for RNA tracking, FLURIL tagging can be applied to internal, genetically encoded URIL RNA sites with minimal structural perturbation, co-expression of protein-fusion labels or significant increase in molecular weight and steric bulk. We demonstrate effective FLURIL tagging of intracellular (HEK-293) RNAs, ribonucleoprotein (RNP) complexes and live cell (U2-OS) tracking of genomic loci. FLURIL tracking was internally validated by direct comparison with the most widely used live-cell RNA labeling method, MS2-labeling with MCP-HaloTag and Janelia Fluor dyes. In addition, FLURIL-tagging correctly reported on the endogenous RNP in HEK293 cells formed from TAR DNA binding protein 43 (TDP-43-tdTomato) and UG repeat RNA. The FLURIL strategy was also successfully applied to guide RNA (gRNA) in CRISPR-dCas complexes to enable live cell tracking of a low-copy number genomic locus (IDR3), internally benchmarked against MS2/HaloTag labeling of CRISPR-Sirius gRNA targeted to a proximal locus (IDR2). Notably, FLURIL-tagged IDR2 exhibited similar brightness as loci targeted by CRISPR-Sirius gRNA complexes, which bear 8-MS2 hairpins for protein labeling. Together, these experiments show that FLURIL tagging can simply and reliably track intracellular RNA, RNPs, and DNA, with a streamlined molecular footprint relative to other methods. Importantly, these data also indicate that FLURIL tagging is fully compatible with existing labeling methods without crosstalk and may be used to broaden the scope of intracellular RNA and DNA tracking. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/501035v1_figS1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e6cc24org.highwire.dtl.DTLVardef@1646707org.highwire.dtl.DTLVardef@a80268org.highwire.dtl.DTLVardef@363193_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO FLURIL-tagging of RNAs with bPNA probes. (a) Triplex hybridization of a U-rich internal loop (URIL) with bPNA (blue) via base triple formation between the melamine base (M) and two uracil bases (inset). (b) General schematic of labeling strategy described herein. An RNA of interest is engineered to contain an URIL and expressed within the cell, with a fluorogenic bPNA probe introduced via cell culture media. Successful URIL targeting is reported by an increase in emission (green) and confirmed by a previously established RNA binding protein with a fluorescent protein (red) fusion. C_FIG

cell biology↗

bioRxiv2021

Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

microbiology↗

bioRxiv2020

Nanotrap(R) particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free medium methods

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.

microbiology↗

bioRxiv2019

Active monomer RTR-1 derived from the root of Rhodomyrtus tomentosa induces apoptosis in gastric carcinoma cells by inducing ER stress and inhibiting the STAT3 signalling pathway

ObjectiveRhodomyrtus tomentosa, a flowering plant belonging to the Myrtaceae family, is considered an antitumour substance with versatile biological and pharmacological activities. And RTR-1 is an active monomer purified from the root of Rhodomyrtus tomentosa. However, the detail of the mechanisms of anti-cancer activity of RTR-1 remains to be elucidated and the effect on gastric cancer cells is unknown. MethodsCell proliferation was determined by MTT and clone formation assay. The effect of RTR-1 on cell cycle and apoptosis was analyzed utilizing flow cytometry, respectively. Moreover, western blotting was used to detect the expression of cell cycle- and apoptosis-related protein. ResultsBased on MTT and clone formation assay, we noticed that RTR-1 inhibited the proliferation of gastric carcinoma (BGC823 and SGC7901) cells in a dose- and time-dependent manner. Furthermore, the results of flow cytometry and western blotting showed that RTR-1 induced cell cycle arrest in the G2/M phase through the ATM-Chk2-p53-p21 signalling pathway and induced cell apoptosis by inhibiting the signal transducers and activators of transcription 3 (STAT3) pathway and activating the endoplasmic reticulum stress (ER stress) pathway. ConclusionTaken together, these results demonstrate that RTR-1 induces cell cycle arrest and promotes apoptosis in gastric carcinoma, indicating its potential application for gastric cancer therapy.

cancer biology↗