Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses.

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research

Molecular Basis of Inhibitory Modulation of P2X2 Receptors by Zinc and Copper.

P2X receptors are ion channels gated by ATP and modulated by the trace metals zinc and copper. Rat P2X2 (rP2X2) receptors show potentiation in the presence of a submaximal concentration of ATP but are inhibited by zinc at concentrations above 100 µM while human P2X2 (hP2X2) receptors are strongly inhibited by zinc over the range of 2-100 µM. Copper and zinc are released from the presynaptic terminals of some neurons but biological role of zinc modulation of P2X2 receptors is unknown in either species. In Chapter 2, I used the known 3D crystal structure of the zebrafish P2X4.1 receptor as a template to identify candidate residues that might be involved in zinc inhibition of hP2X2 receptors, and then tested the effect of these residues using site directed mutagenesis followed by biochemical and electrophysiological analysis. I demonstrated that a cluster of three histidine residues at the subunit interface controls zinc modulation. I also showed that the low affinity zinc inhibition of rP2X2 receptors can be converted to high affinity inhibition by a single residue change. I explored whether the zinc binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In Chapter 3, I showed that hP2X2 receptors are potently inhibited by copper. This high affinity inhibition has an extremely slow recovery after washout of copper. ATP was required for copper to reach its inhibitory binding site but was not essential for copper to leave the site. The first six cysteines were not required for normal copper inhibition. The same three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Furthermore, DTT, a reducing agent, dramatically accelerated recovery from copper inhibition. Humans with Wilson’s disease have excess amounts of copper accumulation in the brain. The copper sensitivity of hP2X2 suggests that these patients have non-functional P2X2 receptors.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94075/1/sukpunth_1.pd

Photon-directed multiplexed enzymatic DNA synthesis for molecular digital data storage

Writing data in DNA is still a bottleneck due to the reliance on chemical synthesis methods. Here the authors report multiplexed enzymatic DNA synthesis using maskless photolithography

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale