scholarly journals A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

Research ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yi-Lun Ying ◽  
Jie Yang ◽  
Fu-Na Meng ◽  
Shuang Li ◽  
Meng-Ying Li ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Single Molecule ◽  
Critical Role ◽  
Label Free ◽  
Peptide Substrates ◽  
Regulatory Pathways ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
One Step ◽  
Targeted Drug Discovery

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

scholarly journals Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

2018 ◽  
Vol 114 (3) ◽  
pp. 381a
Author(s):  
Nikolas Hundt ◽  
Andrew Tyler ◽  
Gavin Young ◽  
Daniel Cole ◽  
Adam J. Fineberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Label Free ◽  
Actin Nucleation ◽  
Single Molecule Level

scholarly journals Single-molecule nanopore sensing of actin dynamics and drug binding

2019 ◽  
Author(s):  
Xiaoyi Wang ◽  
Mark D. Wilkinson ◽  
Xiaoyan Lin ◽  
Ren Ren ◽  
Keith Willison ◽  
...  
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Eukaryotic Cell ◽  
Molecular State ◽  
Drug Binding ◽  
Actin Dynamics ◽  
Label Free ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Voltage Dependent

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

scholarly journals Nanopore microscope identifies RNA isoforms with structural colors

2021 ◽  
Author(s):  
Filip N Boskovic ◽  
Ulrich Felix Keyser
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Rna Structures ◽  
Transcript Isoforms ◽  
Single Molecule Level ◽  
Structural Colors ◽  
Rna Targets ◽  
Dna Strands ◽  
One Step ◽  
Simultaneous Identification

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers (IDs) with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colors. Structural colors consist of DNA structures, protein labels, native RNA structures, or a combination of all three. The sequence of structural colors of RNA IDs enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one step, enzyme-free reaction in a complex human transcriptome using single-molecule readout.

scholarly journals Quantifying protein-protein interactions by molecular counting with mass photometry

2020 ◽  
Author(s):  
Fabian Soltermann ◽  
Eric D.B. Foley ◽  
Veronica Pagnoni ◽  
Martin R. Galpin ◽  
Justin L.P. Benesch ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Single Molecule ◽  
Protein Interactions ◽  
Analytical Techniques ◽  
Binding Affinities ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Single Molecule Level ◽  
Minimal Invasiveness ◽  
Universal Approach

AbstractInteractions between biomolecules control the processes of life in health, and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are particular desirable because of their simplicity and minimal invasiveness, but struggle to quantify tight interactions. Here, we show that we can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different un-labelled biomolecules and their complexes in mixtures at the single-molecule level by mass photometry. These measurements enable us to quantify binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as to determine the associated kinetics. Our results introduce mass photometry as a rapid, simple and label-free method for studying sub-μM binding affinities, with potential to be extended towards a universal approach for characterising complex biomolecular interactions.

scholarly journals Cellular Lensing and Near Infrared Fluorescent Nanosensor Arrays to Enable Chemical Efflux Cytometry

2020 ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Near Infrared ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Chemical Cytometry

Abstract Nanosensor have proven to be powerful tools to monitor single biological cells and organisms, achieving spatial and temporal precision even at the single molecule level. However, there has not been a way of extending this approach to statistically relevant numbers of living cells and organisms. Herein, we design and fabricate a high throughput nanosensor array in a microfluidic channel that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). An array of nIR fluorescent single walled carbon nanotube (SWNT) nanosensors is integrated along a microfluidic channel through which a population of flowing cells is guided. We show that one can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR emission profiles and extract rich information on a per cell basis at high throughput. This unique biophotonic waveguide allows for quantified cross-correlation of the biomolecular information with physical properties such as cellular diameter, refractive index (RI), and eccentricity and creates a label-free chemical cytometer for the measurement of cellular heterogeneity with unprecedented precision. As an example, the NCC can profile the immune response heterogeneities of distinct human monocyte populations at attomolar (10-18 moles) sensitivity in a completely non-destructive and real-time manner with a rate of ~100 cells/frame, highest range demonstrated to date for state of the art chemical cytometry. We demonstrate distinct H2O2 efflux heterogeneities between 330 and 624 attomole/cell·min with cell projected areas between 271 and 263 µm2, eccentricity values between 0.405 and 0.363 and RI values between 1.383 and 1.377 for non-activated and activated human monocytes, respectively. Hence, we show that our nanotechnology based biophotonic cytometer has significant potential and versatility to answer important questions and provide new insight in immunology, cell manufacturing and biopharmaceutical research.

scholarly journals A label-free approach to detect ligand binding to cell surface proteins in real time

2018 ◽  
Author(s):  
Verena Burtscher ◽  
Matej Hotka ◽  
Yang Li ◽  
Michael Freissmuth ◽  
Walter Sandtner
Keyword(s):  
Ligand Binding ◽  
Real Time ◽  
Single Molecule ◽  
Membrane Capacitance ◽  
Surface Proteins ◽  
Displacement Current ◽  
High Temporal Resolution ◽  
Label Free ◽  
Single Molecule Level ◽  
Electrophysiological Recordings

AbstractElectrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

scholarly journals KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell

2021 ◽  
Vol 7 (6) ◽  
pp. eabd2054
Author(s):  
Nishaben M. Patel ◽  
Meenakshi Sundaram Aravintha Siva ◽  
Ruchi Kumari ◽  
Dipeshwari J. Shewale ◽  
Ashim Rai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Regulatory Mechanism ◽  
Coiled Coil ◽  
Recycling Endosomes ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Membrane Tubulation ◽  
Guanosine Triphosphatase ◽  
Neck Coil ◽  
Unique Mechanism

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil–coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

scholarly journals Studies of bacterial topoisomerases I and III at the single-molecule level

2013 ◽  
Vol 41 (2) ◽  
pp. 571-575 ◽  
Author(s):  
Ksenia Terekhova ◽  
John F. Marko ◽  
Alfonso Mondragón
Keyword(s):  
Single Molecule ◽  
Topoisomerase I ◽  
Dna Topology ◽  
Biological Molecules ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Topoisomerase Iii ◽  
Type Ia ◽  
Dna Strand

Topoisomerases are the enzymes responsible for maintaining the supercoiled state of DNA in the cell and also for many other DNA-topology-associated reactions. Type IA enzymes alter DNA topology by breaking one DNA strand and passing another strand or strands through the break. Although all type IA topoisomerases are related at the sequence, structure and mechanism levels, different type IA enzymes do not participate in the same cellular processes. We have studied the mechanism of DNA relaxation by Escherichia coli topoisomerases I and III using single-molecule techniques to understand their dissimilarities. Our experiments show important differences at the single-molecule level, while also recovering the results from bulk experiments. Overall, topoisomerase III relaxes DNA using fast processive runs followed by long pauses, whereas topoisomerase I relaxes DNA through slow processive runs followed by short pauses. These two properties combined give rise to the overall relaxation rate, which is higher for topoisomerase I than for topoisomerase III, as expected from many biochemical observations. The results help us to understand better the role of these two topoisomerases in the cell and also serve to illustrate the power of single-molecule experiments to uncover new functional characteristics of biological molecules.

TPIP: a novel phosphoinositide 3-phosphatase

2001 ◽  
Vol 360 (2) ◽  
pp. 277-283 ◽  
Author(s):  
Steven M. WALKER ◽  
C. Peter DOWNES ◽  
Nick R. LESLIE
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatase Activity ◽  
Splice Variants ◽  
Critical Role ◽  
Tumour Suppressor ◽  
Metabolizing Enzymes ◽  
Cellular Processes ◽  
Lipid Phosphatase ◽  
Phosphoinositide 3 Kinase ◽  
Phosphatase And Tensin Homologue

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIPα and TPIPβ, appear to be functionally distinct. TPIPα displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P3, PtdIns(3,5)P2, PtdIns(3,4)P2 and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIPβ, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIPα plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues.

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