Mostly natural sequencing-by-synthesis for scRNA-seq using Ultima sequencing.

Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5' and 3' scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.

Multimodal Measurements of Single-Molecule Dynamics Using FluoRBT.

Single-molecule methods provide direct measurements of macromolecular dynamics, but are limited by the number of degrees of freedom that can be followed at one time. High-resolution rotor bead tracking (RBT) measures DNA torque, twist, and extension, and can be used to characterize the structural dynamics of DNA and diverse nucleoprotein complexes. Here, we extend RBT to enable simultaneous monitoring of additional degrees of freedom.

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques.

Torque spectroscopy of DNA: base-pair stability, boundary effects, backbending, and breathing dynamics.

Changes in global DNA linking number can be accommodated by localized changes in helical structure. We have used single-molecule torque measurements to investigate sequence-specific strand separation and Z-DNA formation. By controlling the boundary conditions at the edges of sequences of interest, we have confirmed theoretical predictions of distinctive boundary-dependent backbending patterns in torque-twist relationships.

Recent developments in single-molecule DNA mechanics.

Over the past two decades, measurements on individual stretched and twisted DNA molecules have helped define the basic elastic properties of the double helix and enabled real-time functional assays of DNA-associated molecular machines. Recently, new magnetic tweezers approaches for simultaneously measuring freely fluctuating twist and extension have begun to shed light on the structural dynamics of large nucleoprotein complexes. Related technical advances have facilitated direct measurements of DNA torque, contributing to a better understanding of abrupt structural transitions in mechanically stressed DNA.

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA.

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution.

The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific readout of the minor groove.

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2.

Increase in backbone mobility of the VTS1p-SAM domain on binding to SRE-RNA.

The sterile alpha motif (SAM) domain of VTS1p, a posttranscriptional gene regulator, belongs to a family of SAM domains conserved from yeast to humans. Even though SAM domains were originally classified as protein-protein interaction domains, recently, it was shown that the yeast VTS1p-SAM and the SAM domain of its Drosophila homolog Smaug can specifically recognize RNA hairpins termed Smaug recognition element (SRE). Structural studies of the SRE-RNA complex of VTS1p-SAM revealed that the SAM domain primarily recognizes the shape of the RNA fold induced by the Watson-Crick base-pairing in the RNA pentaloop.

Changes in dynamics of SRE-RNA on binding to the VTS1p-SAM domain studied by 13C NMR relaxation.

RNA recognition by proteins is often accompanied by significant changes in RNA dynamics in addition to conformational changes. However, there are very few studies which characterize the changes in molecular motions in RNA on protein binding. We present a quantitative (13)C NMR relaxation study of the changes in RNA dynamics in the pico-nanosecond time scale and micro-millisecond time scale resulting from interaction of the stem-loop SRE-RNA with the VTS1p-SAM domain.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA.

Solving the structure of PTB in complex with pyrimidine tracts: an NMR study of protein-RNA complexes of weak affinities.

NMR spectroscopy has proven to be a powerful tool for the structure determination of protein/RNA complexes. However, the quality of these structures depends critically on the number of unambiguous intermolecular and intra-RNA nuclear Overhauser effect (NOE) constraints that can be derived. This number is often limited due to exchange phenomena that can cause signal line broadening and the fact that unambiguous NOE assignments are challenging in systems that exchange between different conformations in the intermediate to fast exchange limit.

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied.

Shape-specific recognition in the structure of the Vts1p SAM domain with RNA.

Although the abundant sterile alpha motif (SAM) domain was originally classified as a protein-protein interaction domain, it has recently been shown that certain SAM domains have the ability to bind RNA, defining a new type of post-transcriptional gene regulator. To further understand the function of SAM-RNA recognition, we determined the solution structures of the SAM domain of the Saccharomyces cerevisiae Vts1p (Vts1p-SAM) and the Smaug response element (SRE) stem-loop RNA as a complex and in isolation. The structures show that Vts1p-SAM recognizes predominantly the shape of the SRE rather than its sequence, with the exception of a G located at the tip of the pentaloop.

Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling.

The polypyrimidine tract binding protein (PTB) is a 58 kDa protein involved in many aspects of RNA metabolism. In this study, we focused our attention on the structure of the two C-terminal RNA recognition motifs (RRM3 and RRM4) of PTB. In a previous study, it was found that the two RRMs are independent in the free state.

Structure of PTB bound to RNA: specific binding and implications for splicing regulation.

The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity.