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.

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.

Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases.

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.

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

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease-of-readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a parallelized manner. Here, we report a multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with video game music, which is equivalent to 84 trits or 110 bits of data.

A single combination gene therapy treats multiple age-related diseases.

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-ß receptor 2 [sTGFßR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

Meta-omics analysis of elite athletes identifies a performance-enhancing microbe that functions via lactate metabolism.

The human gut microbiome is linked to many states of human health and disease1. The metabolic repertoire of the gut microbiome is vast, but the health implications of these bacterial pathways are poorly understood. In this study, we identify a link between members of the genus Veillonella and exercise performance. We observed an increase in Veillonella relative abundance in marathon runners postmarathon and isolated a strain of Veillonella atypica from stool samples. Inoculation of this strain into mice significantly increased exhaustive treadmill run time. Veillonella utilize lactate as their sole carbon source, which prompted us to perform a shotgun metagenomic analysis in a cohort of elite athletes, finding that every gene in a major pathway metabolizing lactate to propionate is at higher relative abundance postexercise. Using 13C3-labeled lactate in mice, we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill run time performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance.

Potent and long-lasting inhibition of human P2X2 receptors by copper.

P2X receptors are ion channels gated by ATP. In rodents these channels are modulated by zinc and copper. Zinc is co-released with neurotransmitter at some synapses and can modulate neuronal activity, but the role of copper in the brain is unclear. Rat P2X2 receptors show potentiation by 2-100 µM zinc or copper in the presence of a submaximal concentration of ATP but are inhibited by zinc or copper at concentrations above 100 µM. In contrast, human P2X2 (hP2X2) receptors show no potentiation and are strongly inhibited by zinc over the range of 2-100 µM. The effect of copper on hP2X2 is of interest because there are human brain disorders in which copper concentration is altered. We found that hP2X2 receptors are potently inhibited by copper (IC50 = 40 nM). ATP responsiveness recovered extremely slowly after copper washout, with full recovery requiring over 1 h. ATP binding facilitated copper binding but not unbinding from this inhibitory site. A mutant receptor in which the first six extracellular cysteines were deleted, C(1-6)S, showed normal copper inhibition, however reducing agents dramatically accelerated recovery from copper inhibition in wild type hP2X2 and the C(1-6)S mutant, indicating that the final two disulfide bonds are required to maintain the high affinity copper binding site. Three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Humans with untreated Wilson's disease have excess amounts of copper in the brain. The high copper sensitivity of hP2X2 receptors suggests that they are non-functional in these patients.

High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism.

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2-100 µM, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 µM. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We 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 particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nM) to very low (>100 µM) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation.

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.