Million spot binding array platform for exploring and optimizing multiple simultaneous detection events.

Large-scale, high-throughput specificity assays to characterize binding properties within a competitive and complex environment of potential binder-target pairs remain challenging and cost prohibitive. Barcode cycle sequencing (BCS) is a molecular binding assay for proteins, peptides, and other small molecules that is built on a next-generation sequencing (NGS) chip. BCS uses a binder library and targets labeled with unique DNA barcodes. Upon binding, binder barcodes are ligated to target barcodes and sequenced to identify encoded binding events. For complete details on the use and execution of this protocol, please refer to Hong et al. (2022).

Age-related Huntington's disease progression modeled in directly reprogrammed patient-derived striatal neurons highlights impaired autophagy.

Huntington's disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3' untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs.

ProtSeq: Toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes.

We demonstrate early progress toward constructing a high-throughput, single-molecule protein sequencing technology utilizing barcoded DNA aptamers (binders) to recognize terminal amino acids of peptides (targets) tethered on a next-generation sequencing chip. DNA binders deposit unique, amino acid-identifying barcodes on the chip. The end goal is that, over multiple binding cycles, a sequential chain of DNA barcodes will identify the amino acid sequence of a peptide. Toward this, we demonstrate successful target identification with two sets of target-binder pairs: DNA-DNA and Peptide-Protein. For DNA-DNA binding, we show assembly and sequencing of DNA barcodes over six consecutive binding cycles. Intriguingly, our computational simulation predicts that a small set of semi-selective DNA binders offers significant coverage of the human proteome. Toward this end, we introduce a binder discovery pipeline that ultimately could merge with the chip assay into a technology called ProtSeq, for future high-throughput, single-molecule protein sequencing.

MiR-218 steps down to a threshold of motor impairment.

In this issue of Neuron, Amin et al. (2021) generate genetic tools to titrate down levels of miR-218, a motor neuron-enriched microRNA, in vivo. Varying miR-218 dose alters target selection, results in distinct dose-response curves reflecting 3' UTR features, and reveals a miR-218 threshold below which motor neuron deficits emerge.

Generation of Human Neurons by microRNA-Mediated Direct Conversion of Dermal Fibroblasts.

MicroRNAs (miRNAs), miR-9/9*, and miR-124 (miR-9/9*-124) display fate-reprogramming activities when ectopically expressed in human fibroblasts by erasing the fibroblast identity and evoking a pan-neuronal state. In contrast to induced pluripotent stem cell-derived neurons, miRNA-induced neurons (miNs) retain the biological age of the starting fibroblasts through direct fate conversion and thus provide a human neuron-based platform to study cellular properties inherent in aged neurons and model adult-onset neurodegenerative disorders using patient-derived cells. Furthermore, expression of neuronal subtype-specific transcription factors in conjunction with miR-9/9*-124 guides the miNs to distinct neuronal fates, a feature critical for modeling disorders that affect specific neuronal subtypes. Here, we describe the miR-9/9*-124-based neuronal reprogramming protocols for the generation of several disease-relevant neuronal subtypes: striatal medium spiny neurons, cortical neurons, and spinal cord motor neurons.

Microprocessor Recruitment to Elongating RNA Polymerase II Is Required for Differential Expression of MicroRNAs.

The cellular abundance of mature microRNAs (miRNAs) is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs) into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor's differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha) directly associates with the C-terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb). When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.