Detecting RNA base methylations in single cells by in situ hybridization.

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 ~104-107 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 (m62A, m1G and m3U) 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

α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease.

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

Single-molecule visualization of DNA G-quadruplex formation in live cells.

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.Supported by programme grant funding from Cancer Research UK (C9681/A18618, S.B.) core funding from Cancer Research UK (C14303/A17197, S.B.), a Royal Society University Research Fellowship (UF120277 to S.F.L.), Research Professorship (RP150066 to D.K.), a EPSRC (EP/L027631/1 to D.K.) and a BBSRC David Phillips Fellowship (BB/R011605/1 to M.D.A

Novel techniques for genetic analysis

Genetic analysis involves detection of nucleic acids in a sequence-specific manner. Typically, oligonucleotide probes labelled with fluorescent dyes are used to facilitate detection of their complementary sequences. The development of two fluorescent probe formats, each employing DNA-intercalators as a fluorescence quenchers is described. The first, in which the intercalator 9-amino-6-chloro-2-methoxyacridein is covalently linked to the probe, adjacent to a 5’-fluorophore, increases its fluorescence upon hybridisation to the target sequence, due to interaction of the quencher with the probe/target duplex. The hybridisation specificity of these probes has been demonstrated, culminating in their use in real time PCR. The second format, which involves binding of intercalators to the probe/target duplex from free solution, leads to a decrease in fluorescence upon hybridisation. A range of DNA-binding ligands have been screened for use in this context, leading to the use of ethidium bromide as an intercalating quencher in real time PCR. Efficient hybridisation of labelled oligonucleotide probes to their complementary sequences in PCR products is important for sensitive PCR-based genetic analysis. This can be hindered by competition between the probe and one amplicon strand for the target sequence. Several solutions to this problem are evaluated. Among these, the use of 2’-deoxyinosine-5’-triphosphate (dITP) to produce amplicons with reduced Tm is outlined. Synthetic oligonucleotide probes used for genetic analysis must be obtained in high purity. Two hydrophobic tagging monomers for synthesis of the probes used in the multiplex ligation dependent probe amplification (MLPA) assay have been developed. Introduction of the tags at the 5’-end of synthetic oligonucleotides &gt; 100 nt in length facilitates their purification by RP HPLC.</p

Novel techniques for genetic analysis

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Fluorescence based strategies for genetic analysis

Synthetic chemistry has been central to the design of modern methods of genetic analysis. In this article, we discuss the underlying chemistry and biophysical principles that have been used in the development of robust methods for the analysis of DNA in the diagnostic laboratory

Naphthalenyl- and anthracenyl-ethynyl dT analogues as base discriminating fluorescent nucleosides and intramolecular energy transfer donors in oligonucleotide probes

Fluorescent thymidine analogues functionalised in the 5-position with the moieties naphthalenylethynyl (NeT), anthracenylethynyl (AeT) and anthracenylbuta-1,3-diynyl (AeeT) have been incorporated into oligonucleotides. The modified oligonucleotides undergo significant emission enhancement when hybridised to fully complementary strands and a decrease in fluorescence emission when the modified thymine is paired with guanine. Thus these analogues are potentially useful as base discriminating fluorescent nucleosides (BDFs). When a fluorescein dT monomer is incorporated into the same oligonucleotide strand as the modified base, energy transfer enhances the fluorescein emission, particularly upon duplex formation. These dual-labelled probes may be useful for genetic analysis to detect point mutations and SNPs and could provide multiplexing capability

Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10?12 M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin?biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin?biotin interaction.<br/

Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo [2,3-d] pyrimidin-2(7H)-one nucleoside analogues

Substituted 3H-pyrrolo[2,3-d] pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity compared to T

Super-resolution imaging reveals α-synuclein seeded aggregation in SH-SY5Y cells

Funder: Royal Society; doi: https://doi.org/10.13039/501100000288Funder: UK Dementia Research InstituteAbstract: 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