Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells

CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics

Noncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated with many different diseases, such as cancer, dementia, and cardiovascular conditions. The key for effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. In this work, the first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a‐powered microfluidic, integrated electrochemical biosensor for the on‐site detection of microRNAs is introduced. Through this unique combination, the quantification of the potential tumor markers microRNA miR‐19b and miR‐20a is realized without any nucleic acid amplification. With a readout time of 9 min and an overall process time of less than 4 h, a limit of detection of 10 pm is achieved, using a measuring volume of less than 0.6 µL. Furthermore, the feasibility of the biosensor platform to detect miR‐19b in serum samples of children, suffering from brain cancer, is demonstrated. The validation of the obtained results with a standard quantitative real‐time polymerase chain reaction method shows the ability of the electrochemical CRISPR‐powered system to be a low‐cost, easily scalable, and target amplification‐free tool for nucleic acid based diagnostics

CRISPR/Cas13a‐powered electrochemical microfluidic biosensor for nucleic acid amplification‐free miRNA diagnostics

Noncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated with many different diseases, such as cancer, dementia, and cardiovascular conditions. The key for effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. In this work, the first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a‐powered microfluidic, integrated electrochemical biosensor for the on‐site detection of microRNAs is introduced. Through this unique combination, the quantification of the potential tumor markers microRNA miR‐19b and miR‐20a is realized without any nucleic acid amplification. With a readout time of 9 min and an overall process time of less than 4 h, a limit of detection of 10 pm is achieved, using a measuring volume of less than 0.6 µL. Furthermore, the feasibility of the biosensor platform to detect miR‐19b in serum samples of children, suffering from brain cancer, is demonstrated. The validation of the obtained results with a standard quantitative real‐time polymerase chain reaction method shows the ability of the electrochemical CRISPR‐powered system to be a low‐cost, easily scalable, and target amplification‐free tool for nucleic acid based diagnostics

CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics

© 2020 Elsevier B.V. Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient's sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X (‘X’ highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-min-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17–92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.ISSN:0956-5663ISSN:1873-423

Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells