An automated Bayesian pipeline for rapid analysis of single-molecule binding data.

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

Fluorescence Polarization Control for On-Off Switching of Single Molecules at Cryogenic Temperatures.

Light microscopy, allowing sub-diffraction-limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultraresolution structures, brings highly specific labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples, like whole cells, is described.

Advanced 3D Analysis and Optimization of Single-Molecule FISH in Drosophila Muscle.

Single-molecule fluorescence in situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, the utility of smFISH is hindered in thick tissue sections due to the poor probe penetration of fixed tissue, the inaccessibility of target mRNAs for probe hybridization, high background fluorescence, spherical aberration along the optical axis, and the lack of methods for image segmentation of organelles. Studying mRNA localization in 50 µm thick Drosophila larval muscle sections, these obstacles are overcome using sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3D multiple-organelle segmentation. The latter allows independent thresholds to be assigned to different regions of interest within an image stack. This approach therefore facilitates accurate measurement of mRNA location in thick tissues.

Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow.

A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.

CRISPR-Cas9 nuclear dynamics and target recognition in living cells.

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p.

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼ 200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.