Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Summary

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

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

Single molecule (SM) techniques have greatly developed over the past thirty years to respond to the need of overcoming some of the limitations of traditional, bulk solution measurements ^1-3. The manipulation of single biological molecules has created the opportunity to measure mechanical properties of biopolymers⁴ and control the mechanical parameters of protein-protein⁵ and protein-DNA interactions^6,7. SM fluorescence detection, on the other hand, represents an incredibly versatile tool for studying protein activity in vitro and in vivo, leading to the possibility of localizing and tracking single molecule....

Protocol

1. Optical Tweezers Setup with Nanometer Stability

The experimental setup must provide two optical tweezers with pointing stability at the nanometer level and intensity fluctuations of the trapping laser below 1%. Combination of these conditions will assure nanometer stability of the dumbbell under typical tension (1 pN - few tens of pN), trap stiffness (0.1 pN/nm) and measurement bandwidth (image acquisition rate 20 sec^-1). A scheme of the experimental setup is depicted in Figure 1.

1. Optical tweezers design and construction^15,17:
   1. Mount the experimental apparatus on an optic....

Results

In a successful experiment, one (or more) labeled proteins undergo binding/unbinding and/or monodimensional diffusion along the DNA molecule (Figure 3A). Localization of proteins along the DNA molecule allows the quantification of kinetic parameters as a function of the DNA sequence. When buffer conditions causing 1D diffusion are applied, it is possible to follow protein trajectories, and determine, for example, the diffusion coefficient D_1D.

The precise position o.......

Discussion

In the last decade, single molecule manipulation and imaging techniques have seen great progress in terms of spatial and temporal resolution. The combination of manipulation and imaging techniques is at the base of powerful instruments that now allow the control of the mechanical conditions of a single biological polymer, such as DNA, RNA or cytoskeletal filaments, and the simultaneous localization of single proteins interacting with the same polymer. Controlling the mechanical conditions of the trapped polymer is of par.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Elastomeric isolators	Newport	Newdamp	Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies
Optical isolator	Optics for Research	IO-3-YAG-VHP
Nd:YAG laser, 1,064 nm wavelength	Spectra-Physics	Millennia IR
Acousto-optic deflectors (AODs)	A&A optoelectronic	DTS-XY 250
Direct Digital Synthesizers	Analog Devices
Quadrant detector photodiodes	OSI optoelectronics	SPOT-15-YAG
DIO and FPGA board	National Instruments	NI-PCI-7830R
Halogen lamp	Schott	KL 1500 LCD
Condenser	Olympus	U-AAC 1.4NA Aplanat Apchromat
Objective	Nikon	CFI Plan Apochromat 60x 1.2NA water immersion
532 nm laser	Coherent	Sapphire
CCD 200X and 2000X	Hamamatsu	XC-ST70 CE
Electron-multiplied CCD	Hamamatsu	C9100-13
Piezo stage with nm-accuracy	Physik Instrumente	P-527.2CL
Emission Filter	Chroma Technologies	600/100m
Silica beads (1.54 mm)	Bangs Laboratories	SS04N/5303
Albumin from bovine serum (BSA)	Sigma Aldrich	B4287
Pentyl acetate	Sigma Aldrich	46022	Flammable liquid and vapor (No 1272/2008)
Nitrocellulose	Sigma Aldrich	N8267-5EA	Flammable solid  (No 1272/2008)
Heat block	MPM Instruments Srl	M502-HBD	with 2 removable blocks; preheated at 120 °C
NanoPort assemblies	Upchurch Scientific Inc.	N-333
Polyetheretherketone tubing	Upchurch Scientific Inc.	1535
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
Shut-off valves	Upchurch Scientific, Inc.	P-732
Flangeless fittings	Upchurch Scientific, Inc.	LT-111
Fluorinated ethylene propylene tubing	Upchurch Scientific, Inc.	1549
Two computer-controlled solenoid valves	Clippard, Cincinnati, USA	ET-2-H-M5
Pressure transducer	Druck LTD	PTX 1400
biotin-14-dCTP	Life Technologies	19518-018
Terminal deoxynucleotidyl Transferase (TdT)	Thermoscientific	EP0161
ATTO532 maleimide	Sigma Aldrich	68499
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
Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP)	Sigma Aldrich	C4706
L-Glutathione reduced (GSH)	Sigma Aldrich	G4251	Acute toxicity, Oral (Category 5), H303
Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10 kDa cutoff spin concentrators	Merck Millipore	UFC901024
Streptavidin-coated polystyrene beads 1.87 µm	Spherotech, Inc.	SVP-15-5