Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

RNA molecular recording with an engineered RNA deaminase.

RNA deaminases are powerful tools for base editing and RNA molecular recording. However, the enzymes used in currently available RNA molecular recorders such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We designed a platform for directed evolution of RNA molecular recorders. We engineered an RNA A-to-I deaminase (an RNA adenosine base editor, rABE) that has high activity, low bias and low background. Using rABE, we present REMORA (RNA-encoded molecular recording in adenosines), wherein deamination by rABE writes a molecular record of RNA-protein interactions. By combining rABE with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal RNA molecular recording of mammalian Pumilio proteins PUM1 and PUM2 shows that PUM1 competes with PUM2 for a subset of sites in cells. Furthermore, we identify transcript isoform-specific RNA-protein interactions driven by isoform changes distal to the binding site. The genetically encodable RNA deaminase rABE enables single-molecule identification of RNA-protein interactions with cell type specificity.

Nucleosome density shapes kilobase-scale regulation by a mammalian chromatin remodeler.

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.