Nanopore signal deviations from pseudouridine modifications in RNA are sequence-specific: quantification requires dedicated synthetic controls.

Chemical modifications to mRNA respond dynamically to environmental cues and are important modulators of gene expression. Nanopore direct RNA sequencing has been applied for assessing the presence of pseudouridine (ψ) modifications through basecalling errors and signal analysis. These approaches strongly depend on the sequence context around the modification, and the occupancies derived from these measurements are not quantitative.

Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis.

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically-derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analyses.

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).

Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis.

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analysis.

Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases.

Click chemistry-based amplification and detection of endogenous RNA and DNA molecules in situ using clampFISH probes.

Direct labeling and measurement of gene expression in single cells show the tremendous variability otherwise hidden in bulk measurements. Single-molecule RNA fluorescence in situ hybridization (FISH) has become a mainstay in laboratories worldwide for measuring gene expression with precision. However, this method remains relatively low throughput because the total fluorescent signal produced is weak and requires long exposure times and high magnification microscopy, which limits the total number of cells sampled in each image.

Synergistic effect of graphene oxide and zoledronic acid for osteoporosis and cancer treatment.

Zoledronic acid (ZOL) is a third generation bisphosphonate which can be used as a drug for the treatment of osteoporosis and metastasis. In this study, graphene oxide (GO) is conjugated with ZOL, and the nanostructured material is evaluated in terms viability, proliferation and differentiation. Furthermore, the associated morphological changes of bone marrow-derived mesenchymal stem cells (BM-MSC), and Michigan Cancer Foundation-7 (MCF-7) breast cancer cells, as well as the effect of the drugs on mineralization of BM-MSCs are investigated using a variety of characterization techniques including Fourier Transform Infrared Spectroscopy (FTIR), scanning electron microscopy (SEM) as well as alamar blue, acridine orange, and alizarin red assays.