Ved at kombinere Single-molekyle manipulation og Imaging for Studiet af protein-DNA interaktioner
Summary
Her beskriver vi instrumentering og metoder til påvisning af en enkelt fluorescens-mærkede proteinmolekyler, der interagerer med en enkelt DNA-molekyle udspændt mellem to optisk indespærrede mikrosfærer.
Abstract
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
Introduction
Enkelt molekyle (SM) teknikker i høj grad har udviklet sig over de sidste tredive år for at imødekomme behovet for at overvinde nogle af de begrænsninger af traditionelle, bulkopløsning målinger 1-3. Manipulation af enkelte biologiske molekyler er skabt mulighed for at måle mekaniske egenskaber af biopolymerer 4 og styre de mekaniske parametre for protein-protein-5 og protein-DNA-interaktioner 6,7. SM fluorescenspåvisning, på den anden side udgør et utroligt alsidigt værktøj til at studere protein-aktivitet in vitro og in vivo, hvilket fører til muligheden for at lokalisere og spore enkelte molekyler med nanometer præcision. Ved montering af instrumentet point-spread funktion SM billedet, i virkeligheden, kan man udføre lokalisering med en præcision hovedsageligt afhængigt signal-støj-forhold (SNR) og nå en grænse på omkring en nanometer 8,9. Disse metoder finder magtfuldeanvendelser i studiet af dynamikken i motordrevne proteiner, såvel som af de diffusionsprocesser underliggende søgemål i DNA-bindende proteiner. Evnen til at bestemme diffusionskonstanter som en funktion af DNA-sekvensen, opholdstid på målet og nøjagtig måling af DNA længde undersøgt i endimensionale diffusion begivenheder udgør et effektivt værktøj til undersøgelse af protein-DNA-vekselvirkninger dynamik og for undersøgelsen af mekanismerne i specifikke mål søgning.
For nylig er kombination af disse to teknikker fremstilles en ny generation af forsøgsopstillinger 10-14 muliggør samtidig manipulation af et biologisk substrat (for eksempel en actin filament eller et DNA-molekyle) og påvisning / lokalisering af et interagerende partner enzym (for eksempel myosin eller et DNA-bindende protein). Fordelene ved disse teknikker primært hviler på muligheden for at udøve mekanisk kontrol over de fangne polymer således enabling studiet af interaktion dynamik versus kræfter eller momenter. Også den metode tillader måling af biokemiske reaktioner langt fra overfladen, så man undgår en af de vigtigste begrænsninger klassiske SM metoder, nemlig behovet for immobilisering af molekyler under undersøgelse på en overflade (glas slide eller mikrosfærer).
Kombinationen af to enkelte molekyler teknikker kræver overvindelse flere tekniske vanskeligheder, hovedsagelig som følge af kravene i mekanisk stabilitet og tilstrækkelig SNR (især når de kræver lokalisering med nm præcision) 15. Især når kobling SM fluorescensdetektering med optiske pincet, reduktion af støj og fotoblegning fra fældefangst infrarøde lasere 16 og kontrol af biokemiske buffere til samling af de biologiske komplekser og udførelsen af de eksperimentelle målinger 11 er af allerstørste betydning. Her beskriver vi de metoder for succesfuldmålinger i en dobbelt fældefangst / SM Fluorescens lokalisering setup. Metodikken er illustreret med eksempel laktose repressorprotein (LacI) fluorescensmærket (med Atto532) og påvist, da det binder sig til et DNA-molekyle (fanget mellem to optiske pincet), der indeholder specifikke Laci bindende sekvenser (dvs. operatører). Vi demonstrere effektiviteten af metoden til påvisning af binding af LacI til DNA og udbredelse langs dens kontur i målet søgeprocessen. Metoden er anvendelig for enhver kombination af DNA-sekvensen og DNA-bindende protein, samt til andre systemer (mikrotubuli eller actinfilamenter og motoren proteiner, der interagerer med dem).
Protocol
Representative Results
Discussion
I det sidste årti, har enkelt molekyle manipulation og billedbehandling teknikker set store fremskridt i form af rumlige og tidsmæssige opløsning. Kombinationen af manipulering og billeddannende teknikker er ved foden af stærke instrumenter, der nu tillader kontrol af de mekaniske betingelser i en enkelt biologisk polymer, såsom DNA, RNA eller cytoskeletale filamenter, og den samtidige lokalisering af enkelte proteiner interagerer med den samme polymer . Styring de mekaniske fanget polymer er af særlig…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Gijs wuite Erwin JG Peterman, og Peter Gross efter hjælp til mikrofluidik og Alessia Tempestini hjælp til prøveforberedelse. Denne forskning blev finansieret af Den Europæiske Union syvende rammeprogram (FP7 / 2007-2013) under tilskudsaftale n ° 284464 og fra det italienske ministerium for uddannelse, universiteter og forskning FIRB 2011 RBAP11X42L006, Futuro i Ricerca 2013 RBFR13V4M2, og inden for rammerne af flagskibsprojekt Nanomax.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description | Web Address |
| elastomeric isolators | Newport | Newdamp | Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies | http://www.newport.com |
| optical isolator | Optics for Research | IO-3-YAG-VHP | http://www.ofr.com | |
| Nd:YAG laser, 1064 nm wavelength | Spectra-Physics | Millennia IR | http://www.newport.com/ | |
| acousto-optic deflectors (AODs) | A&A optoelectronic | DTS-XY 250 | http://www.aaoptoelectronic.com/ | |
| Direct Digital Synthesizers | Analog Devices | http://www.analog.com/ | ||
| quadrant detector photodiodes | OSI optoelectronics | SPOT-15-YAG | http://www.osioptoelectronics.com | |
| DIO and FPGA board | National Instruments | NI-PCI-7830R | http://www.ni.com | |
| Halogen lamp | Schott | KL 1500 LCD | http://www.schott.com | |
| Condenser | Olympus | U-AAC 1.4NA Aplanat Apchromat | http://www.olympus-global.com/en/ | |
| Objective | Nikon | CFI Plan Apochromat 60x 1.2NA water immersion | http://www.nikoninstruments.com | |
| 532 nm laser | Coherent | Sapphire | http://www.coherent.com | |
| CCD 200X and 2000X | Hamamatsu | XC-ST70 CE | http://www.hamamatsu.com | |
| electron-multiplied CCD | Hamamatsu | C9100-13 | http://www.hamamatsu.com/ | |
| piezo stage with nm-accuracy | Physik Instrumente | P-527.2CL | http://www.physikinstrumente.com/ | |
| Emission Filter | Chroma Technologies | 600/100m | http://www.chroma.com | |
| silica beads (1.54 mm) | Bangs Laboratories | SS04N/5303 | http://www.bangslabs.com/ | |
| Albumin from bovine serum (BSA) | Sigma Aldrich | B4287 | http://www.sigmaaldrich.com/ | |
| pentyl acetate | Sigma Aldrich | 46022 | Flammable liquid and vapour (No 1272/2008) | http://www.sigmaaldrich.com/ |
| nitrocellulose | Sigma Aldrich | N8267-5EA | Flammable solid (No 1272/2008) | http://www.sigmaaldrich.com/ |
| heat block | MPM Instruments Srl | M502-HBD | with 2 removable blocks; preheated at 120° C | http://www.mpminstruments.com |
| NanoPort assemblies | Upchurch Scientific Inc. | N-333 | http://www.upchurch.com/ | |
| polyetheretherketone tubing | Upchurch Scientific Inc. | 1535 | http://www.upchurch.com/ | |
| home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass | ||||
| luer lock-tip syringes 2.5 mL | Terumo | SS 02LZ1 | http://www.terumomedical.com | |
| shut-off valves | Upchurch Scientific, Inc. | P-732 | http://www.upchurch.com/ | |
| flangeless fittings | Upchurch Scientific, Inc. | LT-111 | http://www.upchurch.com/ | |
| fluorinated ethylene propylene tubing | Upchurch Scientific, Inc. | 1549 | http://www.upchurch.com/ | |
| two computer-controlled solenoid valves | Clippard, Cincinnati, USA | ET-2-H-M5 | http://www.clippard.com | |
| pressure transducer | Druck LTD | PTX 1400 | ||
| biotin-14-dCTP | Life Technologies | 19518-018 | http://www.lifetechnologies.com/ | |
| Terminal deoxynucleotidyl Transferase (TdT) | Thermoscientific | EP0161 | http://www.thermoscientificbio.com/ | |
| ATTO532 maleimide | Sigma Aldrich | 68499 | http://www.sigmaaldrich.com/ | |
| N,N-dimethylformamide (DMF) | Sigma Aldrich | 227056 | Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311 | http://www.sigmaaldrich.com/ |
| Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) | Sigma Aldrich | C4706 | http://www.sigmaaldrich.com/ | |
| L-Glutathione reduced (GSH) | Sigma Aldrich | G4251 | Acute toxicity, Oral (Category 5), H303 | http://www.sigmaaldrich.com/ |
| Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10 kDa cutoff spin concentrators | Merck Millipore | UFC901024 | http://www.merckmillipore.it/ | |
| streptavidin-coated polystyrene beads 1,87 µm | Spherotech, Inc. | SVP-15-5 | http://www.spherotech.com/ |
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