Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell ...profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
The analysis of single-molecule fluorescence resonance energy transfer (FRET) trajectories has become one of significant biophysical interest. In deducing the transition rates between various states ...of a system for time-binned data, researchers have relied on simple, but often arbitrary methods of extracting rates from FRET trajectories. Although these methods have proven satisfactory in cases of well-separated, low-noise, two- or three-state systems, they become less reliable when applied to a system of greater complexity. We have developed an analysis scheme that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the most likely time sequence of underlying states for each trajectory. Together with a transition density plot and Bayesian information criterion we can also determine the number of different states present in a system in addition to the state-to-state transition probabilities. Here we present the algorithm and test its limitations with various simulated data and previously reported Holliday junction data. The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct.
The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 ...activity remain ambiguous. In particular, because of the lack of a method with high spatio‐temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single‐molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on‐target sequence. We propose a model in which lateral diffusion competes with three‐dimensional diffusion and thus is involved in PAM finding and consequently on‐target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.
Synopsis
Single‐molecule FRET studies illustrate that lateral diffusion contributes to protospacer adjacent motif (PAM) and target site search by the CRISPR/Cas9 nuclease, expanding the Cas9 search mode beyond random 3D diffusion and emphasizing the importance of neighboring sequences for genetic engineering approaches.
Single‐molecule FRET allows real‐time monitoring of transient Cas9‐DNA interactions.
Weak PAM binding allows Cas9 to probe neighboring sequences by facilitated diffusion.
Cas9 can laterally diffuse between individual target sites.
Multiplicity of PAM sequences can interfere with efficient on‐target binding.
Single‐molecule FRET reveals that lateral diffusion contributes to target search by the CRISPR/Cas9 nuclease and that density of protospacer adjacent motifs (PAM) affects on‐target binding rates.
The precise control of microRNA (miRNA) biogenesis is critical for embryonic development and normal cellular functions, and its dysregulation is often associated with human diseases. Though the birth ...and maturation pathway of miRNA has been established, the regulation and death pathway remains largely unknown. Here, we report the RNA-binding proteins, Lin28a and Lin28b, as posttranscriptional repressors of let-7 miRNA biogenesis. We observe that the Lin28 proteins act mainly in the cytoplasm by inducing uridylation of precursor let-7 (pre-let-7) at its 3′ end. The uridylated pre-let-7 (up-let-7) fails Dicer processing and undergoes degradation. We provide a mechanism for the posttranscriptional regulation of miRNA biogenesis by Lin28 which is highly expressed in undifferentiated cells and certain cancer cells. The Lin28-mediated downregulation of let-7 may play a key role in development, stem cell programming, and tumorigenesis.
CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 ...helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only.
Display omitted
•Cas3 reels target DNA in spring-loaded bursts 3 bp at a time•The 3-bp bursts consist of a kinetic unwinding step size of 1 nt•The nuclease domain of Cas3 is intrinsically inefficient in cleaving DNA•The helicase domain repeatedly presents target DNA to the inefficient nuclease domain.
Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.
Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target ...messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2–4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2–8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.
Display omitted
•Argonaute uses one-dimensional diffusion as a search mechanism•Argonaute initially probes for target sites using a small region (nt 2–4) of miRNA•The seed (nt 2–8) is the minimal motif required for stable binding to target sites•The lateral diffusion promotes cooperativity between neighboring target sites
Argonaute identifies miRNA targets by scanning potential target RNAs using one-dimensional diffusion while probing for sites complementary to a small segment of the miRNA and remains stably associated to sites complementary to the full miRNA seed.
Single-molecule peptide fingerprinting van Ginkel, Jetty; Filius, Mike; Szczepaniak, Malwina ...
Proceedings of the National Academy of Sciences - PNAS,
03/2018, Letnik:
115, Številka:
13
Journal Article
Recenzirano
Odprti dostop
Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. ...However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.
Argonaute proteins use microRNAs (miRNAs) to identify mRNAs targeted for post‐transcriptional repression. Biochemical assays have demonstrated that Argonaute functions by modulating the binding ...properties of its miRNA guide so that pairing to the seed region is exquisitely fast and accurate. However, the mechanisms used by Argonaute to reshape the binding properties of its small RNA guide remain poorly understood. Here, we identify a structural element, α‐helix‐7, in human Argonaute2 (Ago2) that is required for speed and fidelity in binding target RNAs. Biochemical, structural, and single‐molecule data indicate that helix‐7 acts as a molecular wedge that pivots to enforce rapid making and breaking of miRNA:target base pairs in the 3′ half of the seed region. These activities allow Ago2 to rapidly dismiss off‐targets and dynamically search for seed‐matched sites at a rate approaching the limit of diffusion.
Synopsis
The RNA‐Induced Silencing Complex (RISC) is guided to target mRNAs via complementarity to the Argonaute‐bound miRNA. Argonaute2 controls target pairing in the miRNA seed region using a structural element (helix‐7) and thereby enables fast and accurate target searches.
Structural and biochemical data reveal how Ago2 mediates miRNA target search and binding.
The miRNA seed region is composed of two functionally distinct domains.
The seed 5′ domain is immobile and the 3′ domain is flexible and dynamic.
Dynamics of the seed 3′ domain are controlled by helix‐7 in Ago2.
Helix‐7 enables efficient miRNA target searches by accelerating the making and breaking of miRNA:target base pairs and minimizing time spent on off‐targets.
Argonaute2, the key effector of microRNA‐mediated gene silencing, controls base‐pairing in the miRNA seed region to facilitate fast and accurate binding of target mRNAs.
PDF
