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 detection and fluorescent probes. For a single molecule study, it is important to label the protein of interest with a suitable fluorescent probe since fluorescence detection systems are mostly available commercially.

Fluorescent proteins are commonly used in molecular biology. However, the low fluorescent brightness and stability against photobleaching restrict its application in many single molecule assays. Quantum dots (Qdots) are tiny light-emitting nanoparticles⁴. Due to their unique optical properties, Qdots are 10 - 20 times brighter and several thousand times more stable than the widely used organic dyes⁵. Moreover, Qdots have a large Stokes shift (the difference between the position of excitation and emission peaks)⁵. Thus, Qdots can be used for long-time observation (minutes to hours) and acquisition of images with high signal-to-noise ratios, while they cannot be utilized in the quantitative assays.

To date, there are two approaches to label a target protein with Qdots site-specifically: labeling with the aid of Qdot-conjugated primary or secondary antibodies^6,7,8; or labeling the target protein with Qdots directly, which is based on the strong interaction between biotin and streptavidin^{9,10,11,12,13}. Streptavidin-coated Qdots are commercially available. In our recent study, site-specifically biotinylated proteins in budding yeast with high efficiency were purified by co-overexpression of BirA and Avi-tagged proteins in vivo¹⁰. By following and optimizing the single-molecule assays^14,15,16,17, we observed the interactions between Qdot-labeled proteins and DNA at the single molecule level using TIRFM¹⁰.

Here, we choose the budding yeast origin recognition complex (ORC), which can specifically recognize and bind to the autonomously replicating sequence (ARS), as our protein of interest. The following protocol presents a step-by-step procedure of visualizing the interaction of Qdot-labeled ORC with ARS using TIRFM. The preparation of the site-specifically modified DNA substrate, the DNA biotinylation, the coverslip cleaning and functionalization, the flow-cell assembly, and the single-molecule imaging are described.

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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 product, and incubate the reaction at 30 °C for 90 min. Then, add an additional 25 µL of lambda packaging extracts into the reaction tube. Continue to incubate the reaction at 30 °C for 90 min.
   3. Add 500 µL of sterile dilution buffer (10 mM Tris-HCl (pH 8.3), 100 mM NaCl, 10 mM MgCl₂) into the reaction system, and mix gently by turning the tube upside down several times. Add 25 µL of chloroform, mix gently and store at 4 °C.
   4. Add 100 µL of packaged phage and 100 µL of bacteria LE392MP (cultured at 0.8 - 1.0 using LB medium adding with 10 mM MgSO₄) into a new tube. Incubate at 37 °C for 15 minutes.
   5. Add 200 µL of phage-bacterium mixture into 4 mL oftop agar (LB medium + 0.7% agar + 10 mM MgSO₄, cooled to 48 °C). Mix immediately by turning the tube upside down several times and pour it onto a pre-warmed (37 °C) LB plate.
   6. Incubate the plate at 37 °C overnight and screen the λ-ARS317 plaque using PCR and sequencing.
2. DNA substrate purification from liquid lysates^11,18
   1. Pick one plaque into 200 µL of sterile ddH₂O with 10 mM MgCl₂ and 10 nM CaCl₂. Mix with 200 µL of bacteria LE392MP (cultured overnight in LB medium), and incubate at 37 °C for 15 min.
   2. Add the phage-bacterium mixture into 100 mL of NZCYM (10 g/L NZ-amine, 5 g/L yeast extract, 5 g/L NaCl, 1 g/L Casamino Hydrolysate, and 2 g/L MgSO₄·7H₂O) medium and culture for 7 h at 37 °C. Add 250 µL of chloroform into the culture and shake for another 10 min.
   3. Transfer the culture into a 200 mL flask. Add 5.8 g of NaCl (1 M final concentration), stir to dissolve by hand, and incubate on ice for 30 min.
   4. Centrifuge at 12,000 x g for 10 min at 4 °C to remove the cell debris. Collect the supernatant into a 200 mL flask and add 10% PEG₈₀₀₀ (m/V). Stir to dissolve by magnetic stirring apparatus and incubate on ice for 30 min or longer.
   5. Centrifuge at 12,000 x g for 10 min at 4 °C to precipitate the bacteriophage. Remove the supernatant.
   6. Resuspend the precipitate in 2 mL of phage dilution buffer. Transfer it into a 15 mL tube and add 10 µL of RNase (final concentration is 20 µg/mL) and 40 µL of DNase (final concentration is 5 µg/mL). Incubate at 37 °C for 30 min.
   7. Add 2 mL of 0.3 M Tris-HCl (pH 9.0), 100 mM EDTA + 1.25% SDS, 15 µL proteinase K (final concentration is 10 µg/mL). Incubate at 65 °C for 10 min.
   8. Add 2 mL of pre-cooled potassium acetate (3 M, pH 4.8), and incubate the tube on ice for 10 min.
   9. Centrifuge at 8,000 x g for 10 min at 4 °C to remove the insoluble materials.
   10. Add 0.7x volume of isopropanol into the supernatant. Mix by turning the tube upside down several times, and incubate for 2 min at room temperature (RT).
   11. Centrifuge at 8,000 x g for 10 min at RT to precipitate DNA. Remove the supernatant.
   12. Wash the DNA once with 70% ethanol by centrifugation at 8,000 x g for 10 min. Remove the supernatant.
   13. Elute the DNA using 500 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) gently, and transfer the DNA into 1.5 mL tube.
   14. Extract the DNA twice using phenol: chloroform by centrifugation at 8,000 g for 10 min.
   15. Precipitate with 0.7x volume isopropanol. Mix by turning the tube upside down gently, and centrifuge at 8,000 x g for 10 min.
   16. Wash once with 70% ethanol, resuspend in 200 µL of TE. Measure the DNA concentration, and store at -20 °C in 12.5 µg aliquots.

2. λ-ARS317 DNA Biotinylation

1. To phosphorylate biotinylated oligonucleotides (5'-AGGTCGCCGCC-TEG-Biotin-3') complementary to the left end of native λ DNA, add 1 µL (100 µM) of oligonucleotides, 7.5 µL of ddH₂O, 1 µL of 10x ligase buffer, and 0.5 µL of T4 PNK. Incubate the reaction at 37 °C for 3 h.
2. To phosphorylate λ-ARS317, add 12.5 µg of λ-ARS317, 3 µL of 10x ligase buffer, 1 µL of T4 PNK, and ddH₂O to total volume of 30 µL. Incubate the reaction at 37 °C for 3 h.
3. To anneal oligonucleotides with λ-ARS317, add 0.5 µL of phosphorylated oligonucleotides, 195 µL of ddH₂O, 25 µL of 10×ligase buffer into the phosphorylated λ-ARS317 tube. Mix gently by turning the tube upside down and incubate at 65 °C for 5 min. Turn off the block heater, and let the sample cool to below 33 °C in the block.
4. To ligate oligonucleotides with λ-ARS317, add 1 µL of T4 DNA ligase and 0.63 µL of ATP (200 mM). Mix gently by turning the tube upside down and incubate at room temperature for 2 h or 4 °C overnight. Store at 4 °C for 1 month.
   NOTE: To avoid breaking up the DNA, all the mixing steps should be done by gently turning the tube upside down, instead of mixing using pipettes.

3. Coverslip Cleaning and Functionalization

1. Coverslip cleaning
   1. Place 20 coverslips into 4 staining jars (5 coverslips/jar), sonicate for 30 minutes in ethanol, and rinse the coverslips with ultrapure H₂O 3 times.
   2. Sonicate for 30 minutes with 1 M potassium hydroxide (KOH), and rinse the coverslips with ultrapure H₂O 3 times. Repeat the ethanol and KOH sonication once.
   3. Sonicate with acetone for 30 minutes, and then rinse with ultrapure H₂O thoroughly (at least 3 times).
      NOTE: Acetone must be removed thoroughly by rinsing with ultrapure H₂O, because it may cause an explosion when organic solvent (such as acetone) mixed with the piranha solution mistakenly¹⁴.
   4. Place the coverslips into piranha solution (3:1 mixture of H₂SO₄ and 30% H₂O₂) and incubate at 95 °C for 1 h.
      NOTE: Piranha solution is highly energetic and erosive, so use with caution. When preparing piranha solution, add 50 mL of H₂O₂ first into a 500 mL glass beaker, and then add 150 mL of H₂SO₄ slowly into the beaker.
   5. Rinse the coverslips with ultrapure H₂O 5 times. Then wash each coverslip with ultrapure H₂O thoroughly using 3 beakers filled with ultrapure H₂O, and dry the coverslip using paper from the edge of the coverslip. Place the coverslip into the staining jar and dry the coverslip thoroughly in a 110 °C oven for 30 minutes.
      1. Rinse the coverslip with methanol and place the staining jar into the 110 °C oven again to dry the coverslip thoroughly.
         NOTE: Drying the coverslip thoroughly is very important, because APTES will have lots of possible surface structures once it is in the presence of water¹⁹.
2. Coverslip functionalization
   1. Add 70 mL of silane solution (93% methanol, 5% acetic, and 2% APTES) into each jar, screw the cap and leave the jar at room temperature overnight.
   2. Wash and dry the coverslips as described in step 3.1.5, except dry the coverslips thoroughly using nitrogen gas instead of placing them in the oven.
   3. Dissolve 150 mg of mPEG (methoxy-polyethylene glycol) and 6 mg of biotin-PEG (biotin-polyethylene glycol) into 1 mL of 0.1 M fresh-made NaHCO₃ (pH 8.2) thoroughly. Then centrifuge at 17, 000 x g for 1 min to remove insoluble PEGs.
      NOTE: Freshly made NaHCO₃ is ready; there is no need to adjust its pH.
   4. Place the silanized coverslips in boxes, and put two small coverslips on the top of two ends of the silanized coverslips. Pipette 100 µL of PEG solution on the center of the silanized coverslip, and place another silanized coverslip on the top.
   5. Add some ultrapure H₂O in the box to keep it humid, and incubate the coverslips with PEG solution for at least 3 h in the dark. An overnight incubation can work as well.
   6. Separate the coverslip pairs and keep the functionalized surface face up, rinse the coverslips using ultrapure H₂O extensively, and dry them with nitrogen gas.
   7. Mark the functionalized side of the coverslips on one of the corners using a marker pen, keep the functionalized side face up, place them in boxes and store the boxes in the vacuum desiccator for 1 month.
   8. To store the coverslips for a longer time, place one coverslip into a 50 mL tube drilled with one hole on its cap, then put the tube into a plastic bag, and seal the bag using a vacuum sealer. In this way, the coverslips can be stored at -20 °C about 3 months.

4. Flow Cell Assembly

1. Cut a 15 mm × 2 mm channel at the center of a piece of double-sided tape (30 mm × 12 mm) using a puncher.
2. Peel off the paper side of the double-sided tape and paste it on the glass slide with two holes. Press to remove air bubbles. Based on our experience, it is easier to remove air bubbles by peeling off the paper side than the plastic side of the double-sided tape.
3. Cut a functionalized coverslip (60 mm × 24 mm) into four pieces (30 mm × 12 mm) using a diamond-tipped glass scribe, and remove the debris using nitrogen gas. Remember to keep the functionalized side face up.
4. Peel off the plastic side of the double-sided tape, and paste the slide on the functionalized coverslip. Press gently to remove air bubbles between the coverslip and the tape. Removing air bubbles thoroughly can protect the flow cell from leaking while buffers were pumped into it.
5. Insert inlet and outlet tubing into small and big holes, respectively. Fix the tubing using epoxy.
6. Pump 20 µL of streptavidin (0.2 mg/mL) into flow cell using a syringe manually and incubate at RT for 10 min. Then pump blocking buffer¹⁰ into flow cell to replace streptavidin, and keep it at room temperature.

5. Single Molecule Visualization

1. Obtain the focal alignment of red and far-red with A5 on the fluorescence microscope test slide #1 by simultaneous excitation using a 532 nm laser and a 640 nm laser. Produce dual wavelength images with splitting optics (See Table of Materials).
2. Place the flow cell on the microscope and connect its outlet tubing to a longer tubing connecting with a 10 mL spring installed on an automated infusion/withdrawal programmable pump.
   NOTE: Pumping buffers into the flow cell mentioned below means withdrawing buffers from the inlet tubing of the flow cell to the syringe.
3. Pump blocking buffer at 500 µL/min into the flow cell to remove air quickly and ensure that the buffer in the flow cell is unblocked. Then pump the blocking buffer at 200 µL/min into the flow cell and flip the outlet tubing to remove air bubbles from the inlet tubing and the flow cell thoroughly.
   NOTE: All buffers pumped into the flow cell should be degassed in a vacuum desiccator for at least 15 minutes before use.
4. Add 0.5 µL of biotinylated λ-ARS317 DNA into 80 µL of blocking buffer, and pump it into the flow cell at 25 µL/min for 2 min. Then flush λ-ARS317 DNA using 200 µL of blocking buffer at a rate of 50 µL/min.
5. Pump 200 µL of binding buffer¹⁰ at a rate of 50 µL/min to remove the blocking buffer in the flow cell.
6. Add 0.2 µL of streptavidin-coated Qdot₇₀₅ (1 µM) and 0.2 µL of biotinylated ORC (1.2 µM) into a tube, and incubate at room temperature for 5 min. Then add 20 µL of binding buffer into the tube, and place it on ice. The final concentration of ORC-Qdot₇₀₅ is about 10 nM.
7. Add 2 µL of ORC-Qdot₇₀₅ (10 nM), 1 µL of DTT (100 mM), 1 µL of ATP (200 mM) into 96 µL of binding buffer. The final concentration of ORC-Qdot₇₀₅ is 0.2 nM.
8. Pump 20 µL of 0.2 nM ORC-Qdot₇₀₅ at the rate of 10 µL/min into the flow cell. Flush out of excessive ORC-Qdot₇₀₅ using 200 µL of binding buffer at the rate of 100 µL/min. Then pump binding buffer with 30 nM SYTOX Orange into the flow cell to stain DNA substrates at a rate of 100 µL/min.
9. Excite the ORC-Qdot₇₀₅ signal and SYTOX Orange stained DNA signal using a 405 nm laser and 532 nm laser, respectively. Observe the signals simultaneously with 100 µL/min flow using a Quad-band bandpass filter (FF01-446/510/581/703-25) and record the images by EM-CCD with 100 ms per frame. Collect 20 image stacks from different fields.

6. Data Analysis

1. Crop the images using the Fiji software with a new plugin, which is modified by ourselves based on the Image plugin (OI_cut_RGBmerge) developed by Dr. Ron Vale's lab at University of California, San Francisco.
   1. Crop a stack of images (512 × 512 pixels) into two stacks of images (256 × 256 pixels). One is a 532 nm laser exciting result, and the other is a 405 nm laser exciting result.
2. Process 61 sequential images using Fiji-Image-Stacks-Z project (average intensity).
3. Measure the length of DNA (L_DNA) and the distance from the site of ORC-Qdot₇₀₅ (L_ORC) binding on DNA to the DNA's tethered end manually.
4. Calculate the result of L_ORC/L_DNA using excel, analyze the data using the bootstrap method by R, then create the histogram and fit Gaussian distributions.

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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...

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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...

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Materials

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.