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. We produced DRS datasets using modification-free transcripts from in vitro transcription of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analyses of human transcripts.

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

Chemical modifications in mRNAs such as pseudouridine (psi) can regulate gene expression, although our understanding of the functional impact of individual psi modifications, especially in neuronal cells, is limited. We apply nanopore direct RNA sequencing to investigate psi dynamics under cellular perturbations in SH-SY5Y cells. We assign sites to psi synthases using siRNA-based knockdown. A steady-state enzyme-substrate model reveals a strong correlation between psi synthase and mRNA substrate levels and psi modification frequencies. Next, we performed either differentiation or lead-exposure to SH-SY5Y cells and found that, upon lead exposure, not differentiation, the modification frequency is less dependent on enzyme levels suggesting translational control. Finally, we compared the plasticity of psi sites across cellular states and found that plastic sites can be condition-dependent or condition-independent; several of these sites fall within transcripts encoding proteins involved in neuronal processes. Our psi analysis and validation enable investigations into the dynamics and plasticity of RNA modifications.

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

Nanopore direct RNA sequencing (DRS) enables measurements of native RNA modifications. Modification-free transcripts are an important control for DRS. Additionally, it is advantageous to have canonical transcripts from multiple cell lines to better account for human transcriptome variation. Here we generated and analyzed Nanopore DRS datasets for five human cell lines using in vitro transcribed (IVT) RNA. We compared performance statistics amongst biological replicates. We also documented nucleotide and ionic current level variation across cell lines. These data will serve as a resource to the community for RNA modification 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. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand.

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. As such, it is experimentally difficult and time-consuming to sample a large enough population of cells to visualize and quantify specific gene expression of rare cells directly. Several FISH-based tools were recently developed that retain single-molecule sensitivity and specificity while greatly amplifying the fluorescent signal, thus making FISH-based analysis possible using standard microscopes with low magnification objectives. These tools have also enabled the detection of smaller and more specific targets like splice junctions or single nucleotide polymorphisms. Here we will describe one such tool, clampFISH, an oligonucleotide-based fluorescence amplification strategy for visualizing genomic loci and individual RNA transcripts in fixed cells. ClampFISH maintains specificity while amplifying fluorescent signals, making it amenable to high throughput assays such as low magnification microscopy, spatial transcriptomics, and flow sorting. The clampFISH technique involves probing the target RNA or DNA using a series of C-shaped oligonucleotide probes, each with a 3' azide and a 5' alkyne. Hybridization of the probe with the target nucleic acid brings the azide and the alkyne in close proximity, allowing for ligation via bioorthogonal click chemistry (CuAAC). As a result, the probe forms a closed loop around the target sequence, thus enabling stringent washes to remove nonspecific binding in further rounds of amplification and retention of signal throughout liquid handling steps. Iterative rounds of hybridization with C-shaped, fluorescently labeled probes exponentially amplify the fluorescent signal. ClampFISH is simple to implement and expands the utility of in situ hybridization for multiple high throughput techniques such as low magnification microscopy, flow cytometry, and sorting based on RNA expression levels.

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. Nanostructured ZOL-GO with an optimum performance is synthesized using ZOL and GO suspensions with the concentration of 50 µM and 2.91 ng/ml, respectively. ZOL-GO nanostructures can facilitate the mineralization of BM-MSC cells, demonstrated by the formation of clusters around the cells. The results obtained confirm the performance of ZOL-GO nanostructures as promising drug complexes for the treatment of osteoporosis and metastasis.