Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

Summary

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

Abstract

The fluorescence microscopy has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb λ DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified λ DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Introduction

Interactions between protein and DNA are essential to many complex biological processes, such as DNA replication, DNA repair, and transcription. Although conventional approaches have shed light on the properties of these processes, many key mechanisms are still unclear. Recently, with the rapidly developing single molecule techniques, some of the mechanisms have been addressed^1,2,3.

The application of single-molecule fluorescence microscopy on visualizing protein-DNA interactions in real-time mainly depends on the development of fluorescence detect....

Protocol

1. Preparation of λ-ARS317 DNA substrate

1. DNA substrate construction and packaging
   1. Digest native λ DNA using XhoI enzyme; amplify a 543 bp DNA fragment bearing ARS317 from the genomic DNA of budding yeast using primers containing 20 bp homologous sequences of upstream and downstream of XhoI enzyme site on lambda DNA. Add 100 ng of XhoI digested λ DNA and 10 ng of DNA fragment to 10 µL of homologous recombination reaction system, and incubate the reaction at 37 °C for 30 min.
   2. To package the recombination λ DNA, add 25 µL of lambda packaging extracts into the recombination pr....

Results

To visualize the interaction between Qdot-labeled ORC and the ARS, we first constructed the λ-ARS317 DNA substrate. A DNA fragment containing ARS317 was integrated into XhoI site (33.5 kb) of native λ DNA by homologous recombination (Figure 1A). The recombination product was packaged using extracts and the packaged phage particles were cultured on LB plates (Figure 1B). The positive phage plaque was screened by PCR and c.......

Discussion

Here, we present a protocol to observe the interaction between the Qdot-labeled protein and the site-specifically modified λ DNA using the TIRFM in a flow-cell. The necessary steps include site-specific modification of DNA substrate, DNA biotinylation, coverslip cleaning and functionalization, flow-cell preparation, and single-molecule imaging. There are two key points that should be noted. First, all the steps involved with λ DNA should be manipulated gently to decrease any possible damage, e.g., avoi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lambda DNA	New England Biolabs	N3011	Store 25 μL aliquots at -20 ºC.
XhoI enzyme	Thermo Fisher Scientific	FD0694
Quick-fusion cloning kit	Biotool	B22611
MaxPlax Lambda Packaging Extracts	Epicentre	MP5110	Bacterial strain LE392MP is included in this package.
MgSO4	Sinopharm Chemical Reagent Co.,Ltd	10013092	Any brand is acceptable.
Tris	Amresco	0497-5KG
NaCl	Beijing Chemical works	N/A	Any brand is acceptable.
MgCl2	Sinopharm Chemical Reagent Co.,Ltd	10012818
Chloroform	Beijing Chemical works	N/A	Any brand is acceptable.
NZ-amine	Amresco	J853-250G
Casamino acids	Sigma-Aldrich	22090-500G
PEG8000	Beyotime	ST483
Magnetic stirring apparatus	IKA	KMO2 basic
15 mL Eppendorf tube	Eppendorf	30122151	15 mL, sterile, bulk, 500pcs
Rnase	SIGMA	R4875-100MG
Dnase	SIGMA	D5319-500UG
Proteinase K	Amresco	0706-100MG
Biotinylated primers	Thermo Fisher Scientific	N/A
T4 DNA ligase, T4 DNA Ligase, Reaction Buffer (10x)	New England Biolabs	M0202
Coverslip	Thermo Fisher Scientific	22266882
Ethanol	Sinopharm Chemical Reagent Co.,Ltd	10009259
Potassium hydroxide (KOH)	Sigma-Aldrich	306568-100G
Acetone	Thermo Fisher Scientific	A949-4
H2SO4	Sinopharm Chemical Reagent Co.,Ltd	80120891	sulfuric acid
H2O2	Sinopharm Chemical Reagent Co.,Ltd	10011218	30% Hydrogen peroxide
Methanol	Sigma-Aldrich	322415-2L
Acetic acid	Sigma-Aldrich	V900798
APTES	Sigma-Aldrich	A3648
mPEG (methoxy-polyethylene glycol)	Lysan	mPEG-SVA-5000
biotin-PEG (biotin-polyethylene glycol)	Lysan	Biotin-PEG-SVA-5000
NaHCO3	Sigma-Aldrich	31437-500G
Vacuum desiccator	Tianjin Branch Billion Lung Experimental Equipment Co., Ltd.	IPC250-1
Vacuum sealer	MAGIC SEAL	WP300
Diamond-tipped glass scribe	ELECTRON MICROSCOPY SCIENCES	70036
Glass slide	Sail Brand	7101
Inlet tubing	SCI (Scientific Commodties INC.)	BB31695-PE/2	inner diameter 0.38 mm; outer diameter 1.09 mm.
Outlet tubing	SCI (Scientific Commodties INC.)	BB31695-PE/4	inner diameter 0.76 mm; outer diameter 1.22 mm.
Double-sided tape	Sigma-Aldrich	GBL620001-1EA
Epoxy	LEAFTOP	9005	five minutes epoxy
Streptavidin	Sigma-Aldrich	S4762
Fluorescence Microscope	Olympus	IX71
Infusion/withdrawal programmable pump	Harvard apparatus	70-4504
532 nm laser	Coherent	Sapphire-532-50
640 nm laser	Coherent	OBIS-640-100
EMCCD Camera	Andor	DU-897E-CS0-BV
W-View Gemini Imaging splitting optics	Hamamatsu photonics K.K.	A12801-01
TIRF illumination system	Olympus	IX2-RFAEVA2
60×TIRF objective	Olympus	APON60XOTIRF
Quad-edge laser dichroic beamsplitter	Semrock	Di01-R405/488/ 532/635-25x36
Quad-band bandpass filter	Semrock	FF01-446/510/ 581/703-25
Dichroic beamsplitter	Semrock	FF649-Di01-25x36
Emission filter	Chroma Technology Corp	ET585/65m
Emission filter	Chroma Technology Corp	ET665lp
FocalCheck fluorescence, microscope test slide #1	Thermo Fisher Scientific	F36909
SYTOX Orange	Thermo Fisher Scientific	S11368
Qdot705 Streptavidin Conjugate	Thermo Fisher Scientific	Q10163MP	Store at 4 ºC, do not freeze.
ATP	Amresco	0220-25G	Prepare 200 mM ATP solution, using ddH2O, adjust pH to 7.0, and store 10 μL aliquots at -20 ºC.
DTT	Amresco	M109-5G	Prepare 1 M solution using ddH2O, and store 10 μl aliquots at -20 ºC.