A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

The overall goal of this procedure is to build a simple, robust, and high throughput single molecule flow stretching assay for studying transport of molecules along DNA. This is accomplished by first preparing biotin-labeled lambda-DNA by ligating a biotin-labeled oligo to lambda-DNA. The second step is to prepare Polyethylene Glycol or PEG functionalized coverslips by following a one-step reaction protocol.

Next, a high throughput flow cell is constructed by sandwiching a double sided tape with pre-cut channels between a PEG functionalized coverslip and a polydimethylsiloxane or PDMS slab containing inlet and outlet holes. The final step is to track trajectories of single fluorescent molecules in the flow channel by using time-lapse Total Internal Reflection Fluorescence or TIRF imaging. To identify the trajectories of single molecules diffusing along flow stretched lambda-DNA, a reliable and efficient custom single particle tracking software is used to analyze raw fluorescence images.

The main advantages of this assay configuration over alternatives are the reliability of coverslip preparation, higher throughput capacity, reduced hands-on time for sample preparation, and more streamlined unsupervised data analysis. Demonstrating the procedure will be a post-doc in my laboratory, Dr.Kan Xiong. Prior to starting this assay, prepare all necessary reagents as described in the protocol text.

Heat 0.5 milligrams per milliliter lambda-DNA stock to 65 degrees Celsius for 60 seconds and plunge into wet ice right away. Mix 100 microliters of the lambda-DNA solution with two microliters of 10 micromolar biotin-labeled oligo inside a microcentrifuge tube. Heat the mixture at 65 degrees Celsius for 60 seconds and then slowly cool to room temperature.

Place the mixture on ice. Add 11 microliters of T4 DNA ligase reaction buffer and mix gently. Then two microliters equivalent to 800 units of T4 DNA ligase and mix gently.

Incubate the mixture for two hours at 16 degrees Celsius or overnight at four degrees Celsius. Purify the product by using centrifugal filter tubes with a nominal molecular weight limit of 100 kilodaltons. Sonicate number one coverslips in 95%ethanol inside a staining jar for 10 minutes and then rinse with ultrapure water three times.

Fill the staining jar with one molar potassium hydroxide, sonicate for 10 minutes again, and then rinse with ultrapure water three times. Repeat this cycle twice. Dry the coverslips under clean dry nitrogen gas flow.

Further clean and dry the coverslips by conducting air plasma treatment at 900 millitorr pressure for five minutes. Incubate coverslips with 50 microliters of 25 milligrams per milliliter silane-polyethylene glycol-biotin dissolved in 95%ethanol at room temperature for two hours. Wash away excess polyethylene glycol with ultrapure water and dry the polyethylene glycol-coated coverslips under clean dry nitrogen gas flow.

Design flow channels using CAD software and cut the channels on a double sided tape using a tape cutter. Remove tape residuals inside the channels. To make PDMS slabs, thoroughly mix 45 grams of PDMS with five grams of crosslink reagent using a mixer.

Pour the mixture into two 100 millimeter Petri dishes and leave the dishes inside a vacuum chamber for about 30 minutes until all air bubbles are gone. Leave the dishes inside an 80 degree Celsius oven for about two hours until PDMS solidifies. Cut out a PDMS slab that matches the size of the double sided tape with pre-cut channels.

Peel one protection film off the double sided tape and adhere to a flat side of the PDMS slab. Punch outlet and inlet holes on the PDMS slab by using a biopsy hole puncher with a 23 gauge needle. Peel the other protection film off the double sided tape and adhere to the functionalized surface of the coverslip.

Fix the assembled flow cell on a microscope stage insert. Load all pre-degassed reagents into reservoirs that have a Tygon tubing connected to a solenoid valve and another tubing connected to a PEEK tubing that will prevent backflow of reagents. The flow of reagents is controlled by solenoid valves that are controlled by a script programming application.

Prime one flow channel by flowing in blank buffer. Insert three other PEEK tubings into inlet holes. Be careful not to induce air bubble formation inside the channel.

Connect a Tygon tubing with a short PEEK tubing from the outlet hole to a sample waste container. Flow in 0.2 milligrams per milliliter Streptavidin solution and incubate for five minutes. Then flow in blank buffer to wash out the unbound Streptavidin.

Flow in one milligrams per milliliter bovine serum albumin solution and incubate for one minute. Then flow in blank buffer to wash out the unbound bovine serum albumin. Flow in 100 picomolar biotin-lambda-DNA solution and incubate for 10 minutes.

Then flow in blank buffer to wash out the unbound lambda-DNAs. To check the quality of the PEG functionalized coverslip, flow in DNA staining dye such as SYTOX Orange dye and start fluorescence imaging. If the quality of the coverslip is good, the density of flow stretched lambda-DNAs will be high.

Also, tune the TIRF angle to achieve the highest signal-to-noise ratio of images of nearly fully flow stretched lambda-DNAs. To track trajectories of single molecules, start incubations inside a new channel then fuse subnanomolar fluorophore-labeled molecules at a high enough flow rate by using a syringe pump and collect time-lapse fluorescence images with a frame rate of 100 Hertz. Typically 10, 000 frames are collected in one field of view and movies from multiple fields of view are collected.

At the end, flow in DNA staining dye again to confirm that DNAs can still be flow stretched. A custom single particle tracking software will be used to identify trajectories of single molecules diffusing along DNA. The software will first determine the centroid positions of single particles with high accuracies.

Remove particles that are stuck on the coverslip surface and then link particles in different frames to form time-lapse trajectories. From these trajectories, the 1D diffusion constants will be estimated. To start data analysis, open a script programming application and go to the directory of the single particle tracking software.

Open a script named largedataprocess3.m. One important parameter to be defined is the threshold value for single particle detection. To determine the optimal threshold value, run the determine_threshold_value.

m script. This script will visualize how the threshold value affects single particle detection. After determining the optimal threshold value, run the largedataprocess3.

m script. After completion of single particle tracking analysis, the raw trajectories will be filtered by running the trajectory_filtering. m script.

A Graphic User Interface or GUI is built to visualize the filtering step. On the GUI panel, set the minimal displacement along DNA, MinXDisp, to two pixels. Set the maximal displacement transverse to DNA, MaxYDisp, to two pixels.

Set the minimal number of frames, minFrames, to 10. Set the minimal number of states of triplet, minTriplets, to 10. Set the minimal diffusion constant along DNA, D_par to be zero.

Set the maximal estimated diffusion constant transverse to DNA, D_trans, to 10 mega base pair squared per second. Set the minimal statistic parameter, Chi2 Stat, to minus five. Set the minimal ratio of displacement along DNA to that transverse to DNA, MinX/Y, to two.

All raw trajectories that pass these filtering parameters will be listed in table one and those that do not will be listed in table three. Click on a trajectory number in table one. The trajectory will be displayed in graphics one and two.

Click on the play button to play the raw fluorescence images. Click on the add to table2 if this trajectory is a single molecule sliding trajectory. In the end, all trajectories added to table2 will be saved when closing the GUI.

The trajectories that have passed all filtering steps are pooled together from which the mean diffusion constant will be estimated. To calculate the mean diffusion constants, simply run the script calculate_mean_diffusion_constant.m. This procedure will benefit many people studying in vitro single molecule biophysics.