Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing.

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this equilibrium and show that, rather than being driven by the full complex, mRNA binding by eIF3 is instead driven by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in the mRNA recruitment step of translation initiation and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

RNA, a dynamic and flexible molecule with intricate three-dimensional structures, has myriad functions in disease development. Traditional methods, such as X-ray crystallography and nuclear magnetic resonance, face limitations in capturing real-time, single-molecule dynamics crucial for understanding RNA function. This review explores the transformative potential of single-molecule force spectroscopy using optical tweezers, showcasing its capability to directly probe time-dependent structural rearrangements of individual RNA molecules. Optical tweezers offer versatility in exploring diverse conditions, with the potential to provide insights into how environmental changes, ligands and RNA-binding proteins impact RNA behaviour. By enabling real-time observations of large-scale structural dynamics, optical tweezers emerge as an invaluable tool for advancing our comprehension of RNA structure and function. Here, we showcase their application in elucidating the dynamics of RNA elements in virology, such as the pseudoknot governing ribosomal frameshifting in SARS-CoV-2.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson\'s disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.
Pathological protein aggregates in neurodegenerative disorders are difficult to characterise using current methods. We present a novel single‐molecule detection method to specifically detect and characterise α‐synuclein aggregates at picomolar concentrations. We demonstrate the ability to detect aggregates in biologically relevant samples.

Continuous detection of critical markers directly at the point of interest and in undiluted biological fluids represents the next fundamental step in biosensing. The goal of realizing such a platform is utterly challenging because it requires a reversible biosensor that enables the tracking of pico- to nanomolar molecular concentrations over long time spans in a compact device. Here we describe a sensing method based on plasmon-enhanced fluorescence capable of single-molecule detection of unlabeled analyte by employing biofunctionalized gold nanoparticles. The very strong plasmon-enhanced fluorescence signals allow for single-molecule sensing in unaltered biological media, while the use of low-affinity interactions ensures the continuous tracking of increasing and decreasing analyte concentrations with picomolar sensitivity. We demonstrate the use of a sandwich assay for a DNA cancer marker with a limit of detection of picomolar and a time response of 10 min. The enhanced single-molecule signals will allow for miniaturization into a small and cheap platform with multiplexing capability for application in point-of-care diagnostics, monitoring of industrial processes, and safe keeping of the environment.

Calcium ions (Ca2+) reduce NMDA receptor currents through several distinct mechanisms. Among these, calmodulin (CaM)-dependent inhibition (CDI) accomplishes rapid, reversible, and incomplete reduction of the NMDA receptor currents in response to elevations in intracellular Ca2+. Quantitative and mechanistic descriptions of CDI of NMDA receptor-mediated signals have been marred by variability originating, in part, from differences in the conditions and metrics used to evaluate this process across laboratories. Recent ratiometric approaches to measure the magnitude and kinetics of NMDA receptor CDI have facilitated rapid insights into this phenomenon. Notably, the kinetics and magnitude of NMDA receptor CDI depend on the degree of saturation of its CaM binding sites, which represent the bona fide calcium sensor for this type of inhibition, the kinetics and magnitude of the Ca2+ signal, which depends on the biophysical properties of the NMDA receptor or of adjacent Ca2+ sources, and on the relative distribution of Ca2+ sources and CaM molecules. Given that all these factors vary widely during development, across cell types, and with physiological and pathological states, it is important to understand how NMDA receptor CDI develops and how it contributes to signaling in the central nervous system. Here, we review briefly these recent advances and highlight remaining questions about the structural and kinetic mechanisms of NMDA receptor CDI. Given that pathologies can arise from several sources, including mutations in the NMDA receptor and in CaM, understanding how CaM responds to intracellular Ca2+ signals to initiate conformational changes in NMDA receptors, and mapping the structural domains responsible will help to envision novel therapeutic strategies to neuropsychiatric diseases, which presently have limited available treatments.

α-Synuclein (α-Syn) is an intrinsically disordered protein whose aggregation in the brain has been significantly implicated in Parkinson\'s disease (PD). Beyond the brain, oligomers of α-Synuclein are also found in cerebrospinal fluid (CSF) and blood, where the analysis of these aggregates may provide diagnostic routes and enable a better understanding of disease mechanisms. However, detecting α-Syn in CSF and blood is challenging due to its heterogeneous protein size and shape, and low abundance in clinical samples. Nanopore technology offers a promising route for the detection of single proteins in solution; however, the method often lacks the necessary selectivity in complex biofluids, where multiple background biomolecules are present. We address these limitations by developing a strategy that combines nanopore-based sensing with molecular carriers that can specifically capture α-Syn oligomers with sizes of less than 20 nm. We demonstrate that α-Synuclein oligomers can be detected directly in clinical samples, with minimal sample processing, by their ion current characteristics and successfully utilize this technology to differentiate cohorts of PD patients from healthy controls. The measurements indicate that detecting α-Syn oligomers present in CSF may potentially provide valuable insights into the progression and monitoring of Parkinson\'s disease.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are emerging as powerful indicators of diseases. MiRs are secreted in blood plasma and thus may report on systemic aberrations at an early stage via liquid biopsy analysis. We present a method for multiplexed single-molecule detection and quantification of a selected panel of miRs. The proposed assay does not depend on sequencing, requires less than 1 mL of blood, and provides fast results by direct analysis of native, unamplified miRs. This is enabled by a novel combination of compact spectral imaging and a machine learning-based detection scheme that allows simultaneous multiplexed classification of multiple miR targets per sample. The proposed end-to-end pipeline is extremely time efficient and cost-effective. We benchmark our method with synthetic mixtures of three target miRs, showcasing the ability to quantify and distinguish subtle ratio changes between miR targets.

The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.