Hesselberth Lab

Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.Competing Interest StatementL.K.W. has received travel and accommodation expenses from Oxford Nanopore Technologies to present at scientific meetings, and has been a participant in ONT Early Access programs for SQK-RNA004. The remaining authors declare no conflicts of interest.

Antigens from protein subunit vaccination traffic from the tissue to the draining lymph node, either passively via the lymph or carried by dendritic cells at the local injection site. Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens, and archived antigen can be released during subsequent inflammatory stimulus to improve immune responses. Here, we answer questions about how LECs achieve durable antigen archiving and whether there are transcriptional signatures associated with LECs containing high levels of antigen. We used single cell sequencing in dissociated LN tissue to quantify antigen levels in LEC and dendritic cell populations at multiple timepoints after immunization, and used machine learning to define a unique transcriptional program within archiving LECs that can predict LEC archiving capacity in independent data sets. Finally, we validated this modeling, showing we could predict antigen archiving from a transcriptional dataset of CHIKV infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish a unique transcriptional program in LECs that promotes antigen archiving that can be translated to other systems. \#\#\# Competing Interest Statement The authors have declared no competing interest.

Ligation by plant and fungal RNA ligases yields an internal 2'-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA HAC1 The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2'-phosphorylated RNAs during nanopore sequencing. A 2'-phosphate at the splice junction of HAC1 mRNA inhibits 5' $\rightarrow$ 3' degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2'-phosphorylated RNAs on HAC1 and tRNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events in other contexts.

Next generation sequencing (NGS) has provided biologists with an unprecedented view into biological processes and their regulation over the past 2 decades, fueling a wave of development of high throughput methods based on short read DNA and RNA sequencing. For nucleic acid modifications, NGS has been coupled with immunoprecipitation, chemical treatment, enzymatic treatment, and/or the use of reverse transcriptase enzymes with fortuitous activities to enrich for and to identify covalent modifications of RNA and DNA. However, the majority of nucleic acid modifications lack commercial monoclonal antibodies, and mapping techniques that rely on chemical or enzymatic treatments to manipulate modification signatures add additional technical complexities to library preparation. Moreover, such approaches tend to be specific to a single class of RNA or DNA modification, and generate only indirect readouts of modification status. Third generation sequencing technologies such as the commercially available ``long read'' platforms from Pacific Biosciences and Oxford Nanopore Technologies are an attractive alternative for high throughput detection of nucleic acid modifications. While the former can indirectly sense modified nucleotides through changes in the kinetics of reverse transcription reactions, nanopore sequencing can in principle directly detect any nucleic acid modification that produces a signal distortion as the nucleic acid passes through a nanopore sensor embedded within a charged membrane. To date, more than a dozen endogenous DNA and RNA modifications have been interrogated by nanopore sequencing, as well as a number of synthetic nucleic acid modifications used in metabolic labeling, structure probing, and other emerging applications. This review is intended to introduce the reader to nanopore sequencing and key principles underlying its use in direct detection of nucleic acid modifications in unamplified DNA or RNA samples, and outline current approaches for detecting and quantifying nucleic acid modifications by nanopore sequencing. As this technology matures, we anticipate advances in both sequencing chemistry and analysis methods will lead to rapid improvements in the identification and quantification of these epigenetic marks.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.