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

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.


Introduction
A long-standing problem in biology is how endogenous proteins that act at specific sites in the genome can locate their DNA targets quickly enough for the organism to survive and respond effectively to its environment. Studies over the past forty years proposed and largely support the hypothesis that the kinetics of DNA target search by a protein can be accelerated by facilitated diffusion in which the protein alternates between 3D diffusion in the bulk and 1D diffusion (including sliding and hopping processes) along the DNA 1 . It is now known that many proteins involved in gene regulation, nucleic acid metabolism, and other processes are capable of sliding on DNA 2,3,4,5,6,7,8,9 . Furthermore, recent studies reported that even small peptides can bind to and slide on DNA, with the ability to carry a cargo; for example, a protein molecule or PCR primer, along DNA 10,11,12,13,14 .
Over the last 15 years, the single molecule flow-stretching assay has been widely used to study binding and diffusion of molecules along DNA 2,15,16 . In this type of assay, biotinylated double-stranded DNA molecules are immobilized to the surface and a laminar flow is applied to flow stretch the DNA. The > 80% stretched DNAs serve as spatially extended templates for studying binding and transport activity of molecules labeled with fluorophores where the trajectories of single molecules along DNA are tracked by time-lapse fluorescence imaging. In our implementation, optimized for reproducibility and ease of use, this assay consists of five major steps: preparation of biotin-λ-DNA, coverslip functionalization, flow cell construction, fluorescence imaging and data analysis. In previous protocols 17 , glass coverslips were functionalized by first reacting with (3-Aminopropyl)triethoxysilane (APTES) and then with amine-reactive polyethylene glycol (PEG) reagents (e.g., NHS-PEG-biotin) to form a PEG layer that resists nonspecific adsorption of assay components to the coverslip surface. The quality of functionalized coverslips largely depended on the quality of PEG reagents and reaction conditions at each step. Our protocol describes a simplified functionalization protocol and multiplex flow cell construction which requires no liquid adhesive or curing time on the day flow cells are assembled. We also describe a streamlined and robust data analysis procedure 11 that eliminates computationally intensive regression steps by applying a radial symmetry method for centroid localization 18 and a covariance-based diffusion constant estimator 1. Sonicate five no. 1 coverslips in 95% ethanol inside a staining jar for 10 min and then rinse with ultrapure water for three times. NOTE: The fifth coverslip provides a spare in case of breakage. 2. Fill the staining jar with 1 M KOH, sonicate for 10 min and then rinse with ultrapure water three times. 3. Repeat the 2.1-2.2 cycle twice. 4. Dry the coverslips under clean dry N 2 gas flow. 5. Further clean and dry the coverslips by applying air plasma treatment at 900 mTorr pressure for 5 min. 6. Incubate coverslips with 25 mg/mL silane-peg-biotin dissolved in 95% ethanol at room temperature for 2 h. Specifically, sandwich 50 µL silane-peg-biotin solution between two clean dry coverslips, and incubate the coverslip "sandwiches" inside a closed pipette tip box with about 10 mL of water at the bottom (not in contact with the coverslips) to minimize solvent evaporation. 7. Wash away excess PEG molecules with ultrapure water and dry the PEG coated coverslips under clean dry N 2 gas flow.
NOTE: The functionalized coverslips can be stored up to three days under ambient conditions.

Flow cell construction
NOTE: Flow cells are constructed by sandwiching a double-adhesive tape with pre-cut channels between a PEG functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated per flow cell.
1. Design flow channels in a CAD software and cut the channels (the width, depth and length of each channels are 1, 0.13 and 12 mm, respectively) on a double-adhesive tape by using a tape cutter (typically eight channels are integrated per flow cell). Remove tape residuals to form the flow channels. 2. To make PDMS slabs, thoroughly mix 45 g of PDMS with 5 g of crosslinking reagent (enough for making twelve 5-mm thick PMDS slabs which can be stored indefinitely under ambient conditions) using a mixer, pour the mixture into two 10 cm Petri dishes, and leave the dishes inside a vacuum chamber until essentially all air bubbles are gone (manually remove air bubbles if any remain when the dishes are removed from the vacuum chamber). Then transfer the dishes into an 80 °C oven until PDMS solidifies (about 2 h). 3. Cut out a PDMS slab that matches the size of the double-adhesive tape with pre-cut channels. Peel one protection film off the doubleadhesive tape and adhere the film to a flat face of the PDMS slab. Punch the outlet and inlet holes on the PDMS slab using a biopsy holepuncher with a 23 Gauge needle. NOTE: After the DNA is bound to the surface, avoid rapid flows to prevent damage to the tethered DNA molecules.

5.
To check the quality of the functionalized coverslip, flow in DNA staining dye such as Sytox orange under the assay conditions to be used (read 4.6 below), and start fluorescence imaging under 532 nm laser illumination. NOTE: The quality of the functionalized coverslip is typically good and the density of flow stretched DNA molecules will be high. It is essential to have a high enough density of flow stretched DNA molecules so that DNA binding and sliding/1D diffusion events can be observed. 6. Fine tune the TIRF angle to achieve best signal to noise ratio of images of stretched DNA molecules (Figure 1d).  11 . A good negative control is to measure labeled samples of interest in assay buffer in a channel that has no DNA tethered, where no on-DNA diffusion activity should be detected.

Data Analysis:
NOTE: A custom single particle tracking software is used to identify trajectories of single molecules diffusing along DNA and estimate diffusion constants. This software first determines the centroid positions of single particles with high accuracy, identifies particles that are stuck on the coverslip surface, and then links particles in different frames to form time-lapse trajectories. From these trajectories, the 1D diffusion constants are estimated.
1. To start data analysis, open a scripting software (see table of materials) and go to the directory of the single particle tracking software. Open the script named "largedataprocess3.m". One important parameter to be defined is the threshold value for single particle detection. 2. To determine the optimal threshold value, run the "Determine_threshold_value.m" script. This script will indicate how the threshold value affects single particle detection.After determining the optimal threshold value, run the "largedataprocess3.m" script. 3. After completion of single particle tracking analysis, filter the raw trajectories by running the "Trajectory_filtering.m" script.

Discussion
When switching from the traditional two-step coverslip functionalization reaction protocol to a one-step reaction protocol, we noticed that the reliability of coverslip functionalization is significantly improved. As the total hands-on time for coverslip preparation is less than thirty min, we recommend preparing fresh coverslips each day single molecule imaging is planned. To minimize deterioration of silane-PEG-biotin, the reagent should be aliquoted and stored under dry N 2 gas at -20 °C upon receipt. Here we do not use PEG molecules that produce -COO or -CH 3 terminal groups that have been used in the past 20 . The negative charges of -COO groups and the hydrophobic -CH 3 groups were utilized to prevent nonspecific adsorption of DNA molecules and specific protein samples to the surface, respectively. We observe very low surface The shift from glass slide flow cells to PDMS flow cells speeds up the flow cell construction and allows easy integration of multiple channels. We often engineer eight channels per flow cell to enable eight independent experiments per functionalized coverslip. To further increase throughput per coverslip, even larger numbers of micro-fabricated channels could be used.
It is advisable to prevent air bubble formation inside the channel at any point after initial filling. Usually, air bubbles do not cause loss of tethered DNAs (although this is possible), but rather disrupt flow and possibly cause DNAs to stick to the coverslip. Buffer degassing and the addition of small surfactant quantities are typically effective in preventing bubble formation within the tubing and flow channel. In cases where air bubbles are introduced to the tubing, try to flush the bubbles out under a slow flow and make sure that they do not flow through the region where DNAs are tethered.
A streamlined and efficient data analysis software is critical, due to the huge amount of data generated. We usually collect 700,000 highresolution images from measurements on one flow cell, which can be processed using our custom single particle tracking software within one day on a desktop computer with a quad-core processor and 32 Gb memory. The false positive rate of detecting 1D diffusion trajectories by the software (step 5.5) is sample dependent and can be quite low given that: 1) The density of single particles in the FOV is low enough so that there is little ambiguity linking particles across frames (protein samples in general are more prone to nonspecific adsorption to the coverslip than peptide samples, and thus, the buffer and the power density for each protein sample needs to be optimized); 2) The signal to noise ratio of single particles is high enough so that the likelihood of false-identification of particles is low. Still, the software can make occasional mistakes, and we recommend manual inspection of the data using the provided GUI to evaluate the software performance. In an application particularly sensitive to false-positive trajectory detection, process parameters including the threshold value, minFrames, minTriplets, and Chi 2 _stat can be set more stringently at the cost of lower sensitivity for true binding events and potentially greater statistical bias in trajectory identification. We here share and describe our single particle tracking software with the hope of helping standardize data analysis methods and stimulate discussion about best practices.
In our protocol, which requires continuous flow during imaging to maintain the stretched state of the DNA template molecules, the fluid flow could bias the transport of some proteins along DNA templates if they diffuse by a hopping mechanism or if the hydrodynamic radius of the labeled fluorophore is large 22,23 . While such bias can be measured and corrected in most datasets, the transport of proteins labeled with a small fluorophore and diffusing by a sliding mechanism is not typically biased to a detectable extent by flow 2,4,24 . Notably, the impact of the flow velocity on the transport of proteins along DNA has been used to study the mechanisms of protein diffusing along DNA 23 . A closely related alternative approach that enables measurements in the absence of flow utilizes DNA templates with biotin at both termini that are transiently stretched in flow and tethered by both ends to the coverslip such that the DNA molecules remain in an extended configuration when the flow is turned off 25,26,27,28 . The double-tether approach has the advantage of allowing flow-free transport experiments but requires modifying both ends of the DNA template molecules, careful flow control during tethering, and characterizing the distances between the two tethered sites. In addition, the impact of heterogeneous tensions and DNA conformational dynamics across DNA molecules on the single-molecule assay must be assessed.
Altogether, in addition to studying 1D diffusion of molecules along DNA, the as-reported single molecule assay can benefit many in vitro biochemical and biophysical studies at the single molecule level including DNA target search processes by proteins, enzymatic activities on DNA, and more.