Although ordinarily a pivotal tumour-suppressor, p53 can form amyloid aggregates that lose tumour-suppressing functions and exert tumour-promoting activities. Detection of p53 aggregates in liquid biopsies may be a more cost-effective alternative for diagnosis, monitoring and prognostication of glioblastoma (GB) that avoids the logistical challenges of MRI. We have previously developed a novel ultrasensitive immunoassay based on the single-molecule array technology (SiMoA) for detecting p53 aggregates in plasma. Plasma samples were collected intraoperatively from newly diagnosed GB patients during the initial surgery, and from matched control patients undergoing non-cancer surgery. Follow-up samples were collected during outpatient clinics. The assay was used to measure and compare plasma p53 aggregate levels between pre-surgery GB patients and controls. To evaluate the correlation between p53 levels and longitudinal outcome, we identified patients for whom plasma samples were available at =2 timepoints and retrospectively reviewed their health records to map p53 concentrations to onset of treatment (surgery, radiotherapy and/or chemotherapy), survival and tumour recurrence (as indicated by MRI). The assay demonstrated a diagnostic accuracy of 91.48% in distinguishing between GB patients (n=185) and controls (n=35). In our longitudinal review, all patients (n=25) demonstrated clinical congruence with p53 levels either through a rise in p53 levels prior to tumour recurrence (40%), a decline in p53 levels post-treatment (16%), or a combination of these two patterns (44%). Kaplan-Meier curves showed no statistically significant association between p53 levels and survival. Our study highlights the potential utility of plasma p53 aggregate levels for early detection, diagnosis and monitoring of patients with brain cancer. Earlier detection of both primary and recurrent disease in GB would enable rapid institution of treatment thereby maximising the potential for patient benefit.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

We developed the aggregate characterization toolkit (ACT), a fully automated computational suite based on existing and widely used core algorithms to measure the number, size, and permeabilizing activity of recombinant and human-derived aggregates imaged with diffraction-limited and super-resolution microscopy methods at high throughput. We have validated ACT on simulated ground-truth images of aggregates mimicking those from diffraction-limited and super-resolution microscopies and showcased its use in characterizing protein aggregates from Alzheimer's disease. ACT is developed for high-throughput batch processing of images collected from multiple samples and is available as an open-source code. Given its accuracy, speed, and accessibility, ACT is expected to be a fundamental tool in studying human and non-human amyloid intermediates, developing early disease stage diagnostics, and screening for antibodies that bind toxic and heterogeneous human amyloid aggregates.

Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5 h inside HEK cells and 1 day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson’s disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson’s disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

Aggregation of α-synuclein (α-syn) is closely linked to Parkinson’s disease (PD) and the related synucleinopathies. Aggregates spread through the brain during the progression of PD, but the mechanism by which this occurs is still not known. One possibility is a self-propagating, templated-seeding mechanism, but this cannot be established without quantitative information about the efficiencies and rates of the key steps in the cellular process. To address this issue, we imaged the uptake and seeding of unlabeled exogenous α-syn fibrils by SH-SY5Y cells and the resulting secreted aggregates, using super-resolution microscopy. Externally-applied fibrils very inefficiently induced self-assembly of endogenous α-syn in a process accelerated by the proteasome. Seeding resulted in the increased secretion of nanoscopic aggregates (mean 35 nm diameter), of both α-syn and Aβ. Our results suggest that cells respond to seed-induced disruption of protein homeostasis predominantly by secreting nanoscopic aggregates; this mechanism may therefore be an important protective response by cells to protein aggregation.

Antibodies are powerful tools for biological research and clinical applications owing to their ability to bind targets with high affinity and specificity. To exploit them against neurodegeneration, we are developing antibodies to target most toxic early stage aggregates of the protein tau, whose deposition in the brain is linked with Alzheimer's disease and other tauopathies. We generated a panel of single-domain antibodies targeting selected epitopes within tau, using the ‘cascade method’. Briefly, we designed complementary peptides targeting linear epitopes within the sequence of tau using a library of beta-sheet fragments from the protein data bank. We then grafted each peptide into the CDR3 loop of a VH human scaffold. Using this approach, we produced a library of fourteen antibodies covering systematically the length of tau. We will use these antibodies to understand which epitopes are specifically exposed in toxic tau aggregates. In particular, we will employ them in super-resolved imaging, such as DNA points accumulation for imaging in nanoscale topography (DNA PAINT), of protein aggregates. DNA PAINT enables visualization of structures with resolutions below the diffraction limit using short dye-labelled oligonucleotide probes. Transient binding of a dye-labelled ‘imager strand’ to a ‘docking strand’ (DS), which we conjugated to the C-terminus of DesAbs creates a blinking effect. Hence, a highly resolved image can be reconstructed from plotting the measured positions of the hydrolyzed DNA state. Using this technique to image protein aggregates in human cerebrospinal fluid and serum will provide novel insights into the composition, structure, size and number of aggregates present in those samples.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method—methylation-sensitive RNA in situ hybridization (MR-FISH)—that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10 4 –10 7 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m 6 2 A, m 1 G and m 3 U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

The cover feature picture shows how DNA PAINT can be used with aptamers—short sequences of RNA or DNA that can form a 3D structure that can bind to a target molecule—to generate super-resolution images of endogenous amyloid aggregates within patient-derived neuronal cells. The aptamer is modified through the addition of several DNA bases that are complementary to those of a dye-labelled oligonucleotide. A super-resolution image is then formed as the dye-labelled oligonucleotide temporarily hybridises with the aggregate-bound aptamer (bottom left). Super-resolution images of fibrils formed from α-synuclein, the protein associated with Parkinson′s disease, are shown bottom right. More information can be found in the communication by D. Klenerman, M. H. Horrocks et al. on page 2033 in Issue 19, 2018 (DOI: 10.1002/cbic.201800209).

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Oligomers of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrP(C)) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrP(C) and that the species bound to PrP(C) are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAβ-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.

We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10(6) per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

This paper describes a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10-fL droplets at rates of up to 1.3 MHz. The femtodroplets produced with this device encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 minutes of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer down to a concentration of 46 fM.

There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

Single molecule fluorescence spectroscopy (SMFS) has become an established biophysical technique since its first demonstration 35 years ago, along the way stimulating a revolution in the study of DNA sequence, structure and function. In this chapter we summarise the physical and chemical techniques underlying these advances, with emphasis on the selection of fluorophores for single molecule work and their conjugation to DNA. We then highlight a few key applications of SMFS including next generation sequencing technologies, the determination of DNA structures at nanometre resolution and the dissection of protein-DNA interactions one complex at a time.