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.

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.

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.