TITLE: Restriction Endonuclease Protection Assays using Infrared-Fluorescent Probes
AUTHORS: Michael Van Dyke, Isabelle Gracien

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Many proteins sequence-specifically bind duplex DNA, e.g., transcriptional regulatory proteins. Analysis of their interactions can be performed by a variety of methods, including electrophoretic mobility shift assays (EMSA) and quantitative DNase I footprinting. Here we describe an additional electrophoretic method, restriction endonuclease protection assays (REPA), to qualitatively and quantitatively study the interactions of thermophilic transcription regulatory proteins to PCR-generated, infrared-fluorescent DNA probes. REPA utilizes type IIS restriction endonucleases (IISRE), which cleave double-stranded DNA without specificity at a fixed distance from their recognition sequence. Thus, IISREs can be used to probe the occupancy of a suitably situated DNA-binding site for a variety of ligands. REPA has certain advantages as it does not require the maintenance of ligand-DNA complex stability during gel electrophoresis, as is the case with EMSA and is technically far less challenging than quantitative DNase I footprinting.</div></div>

[STEPS]
?. [REPA set up]
Plan REPA reactions. For the example shown here, we investigated the concentration dependence of the Thermus thermophilus HB8 transcriptional regulator SbtR to inhibit IISRE FokI cleavage of an IRD7-labeled (red) test DNA containing the SbtR consensus binding sequence as compared to an IRD8-labeled (green) control DNA. Reactions (10 µL)contained 1 nM each of the test and control DNA and a suitable IISRE cleavage buffer (1x NEB CutSmart®), with the variable being SbtR concentration in the range 1-200 nM. Uncut DNA and FokI cleavage control reactions were also included. Table 1 shows the assembly plan used for this REPA experiment.Table 1. REPA Reaction Plan  ABCDE1SampleExperimentMaster MixSbtR?FokI?2SampleUncut DNA control8 µL2 µL 1x PDB(none)3SampleFokI cleavage control8 µL1 µL 1x PDB1 µL 0.4 U FokI4Sample200 nM SbtR8 µL1 µL 20 µM SbtR1 µL 0.4 U FokI5Sample100 nM SbtR8 µL1 µL 1/2 SbtR1 µL 0.4 U FokI6Sample50 nM SbtR8 µL1 µL 1/4 SbtR1 µL 0.4 U FokI7Sample25 nM SbtR8 µL1 µL 1/8 SbtR1 µL 0.4 U FokI8Sample13 nM SbtR8 µL1 µL 1/16 SbtR1 µL 0.4 U FokI9Sample6.3 nM SbtR8 µL1 µL 1/32 SbtR1 µL 0.4 U FokI10Sample3.1 nM SbtR8 µL1 µL 1/64 SbtR1 µL 0.4 U FokI11Sample1.6 nM SbtR8 µL1 µL 1/128 SbtR1 µL 0.4 U FokI
ABCDE1SampleExperimentMaster MixSbtR?FokI?2SampleUncut DNA control8 µL2 µL 1x PDB(none)3SampleFokI cleavage control8 µL1 µL 1x PDB1 µL 0.4 U FokI4Sample200 nM SbtR8 µL1 µL 20 µM SbtR1 µL 0.4 U FokI5Sample100 nM SbtR8 µL1 µL 1/2 SbtR1 µL 0.4 U FokI6Sample50 nM SbtR8 µL1 µL 1/4 SbtR1 µL 0.4 U FokI7Sample25 nM SbtR8 µL1 µL 1/8 SbtR1 µL 0.4 U FokI8Sample13 nM SbtR8 µL1 µL 1/16 SbtR1 µL 0.4 U FokI9Sample6.3 nM SbtR8 µL1 µL 1/32 SbtR1 µL 0.4 U FokI10Sample3.1 nM SbtR8 µL1 µL 1/64 SbtR1 µL 0.4 U FokI11Sample1.6 nM SbtR8 µL1 µL 1/128 SbtR1 µL 0.4 U FokI
?. [REPA set up]
Assemble REPA reactions. To ensure consistency among reactions, we routinely make a Master Mix containing the constant elements (DNA, buffer, water). Here, our Master Mix contained 52.5 µL water, 10.5 µL 10x NEB CutSmart® buffer, and 10.5 µL each 10 nM IRD7_ST2_SbtR_wt (test) and 10 nM IRD8_trP1_REPSAis (control) DNAs. Note the slight excess of reagents in the prepared Master Mix, to offset any potential pipetting errors. Mixed by brief vortexing and consolidated by brief minifuge centrifugation, 8 µL aliquots of Master Mix were then placed in a series of labeled PCR tubes (1-10) pre-chilled on crushed ice (Figure 3).
?. [REPA execution]
REPA timecourse. REPA experiments consist of the following steps: (A) assemble reactions on ice, (B) protein-DNA binding reaction, (C) sample pre-equilibration for IISRE [if necessary], (D) IISRE cleavage reaction, (E) cleavage reaction stop, (F) native PAGE electrophoresis, (G) imaging by IR fluorescence, and (H) quantitative densitometry. Of these, Steps (B) – (E) are time-dependent. A timeline outlining these steps for the SbtR REPA experiment is shown in Figure 4.
?. [REPA execution]
Protein binding reaction. Before challenge by the IISRE, the protein under investigation needs to be incubated with the test and control DNAs to allow equilibration. As SbtR is a thermophilic protein, incubations were performed at an elevated temperature (55 °C) for 20 min using a digital mini dry bath with a block that accommodates 200 µL PCR tubes.
?. [REPA analysis]
Gel electrophoresis. DNA products in these samples were resolved by native PAGE using hand cast 9.5%:0.5% acrylamide:bisacrylamide, ½x TBE, 10 cm vertical minigels. Electrophoresis was performed initially at 50 V for 5 min, followed by 100 V for 1 h, at which point the Orange G dye had approached the bottom of the gel. A detailed description of our gel electrophoretic technique may be found at https://dx.doi.org/10.17504/protocols.io.mcyc2xw.
?. [REPA analysis]
IR fluorescence imaging. Resolved DNAs were visualized by IR fluorescence using a LI-COR Odyssey Imager and LI-COR Image StudioTM 5.2 software. A general overview may be found at https://dx.doi.org/10.17504/protocols.io.mcyc2xw. Data from our SbtR REPA experiment are shown in Figure 5. Note that as the glass plates used in casting and running the polyacrylamide gel are transparent at near-IR wavelengths. Thus, it is not necessary to remove the gel from these but instead image the entire assembly directly.
?. [REPA set up]
Make serial dilutions of the ligand under investigation. Two-fold serial dilutions of a freshly prepared 20 µM SbtR solution were made in 1x Protein dilution buffer (PDB, 100 mM NaCl, 20 mM Tris-Cl, 0.05% Tween 20, pH 7.8 @ 25 °C), giving the range 16 – 1000 nM SbtR. This was achieved by aliquoting 5 µL 1x PDB in each of seven microwells on a Terasaki plate on ice, adding 5 µL 20 µM SbtR to the rightmost well, and mixing by repeated pipetting (8x). Such was repeated sequentially rightward using just diluted SbtR until the series 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 was made. Afterward, 1 µL of each SbtR dilution was added to the appropriate reaction tube #3 – #10, and the samples mixed by repeated flicking followed by a sharp wrist shake to consolidate the sample in the bottom of the tube. 1x PBD was added to the control reactions #1 and #2, volumes indicated above, and mixed likewise.
?. [REPA set up]
Prepare FokI dilution. Commercial FokI (NEB, typically 4 – 5 U/ µL) was diluted to its optimal concentration for REPA as determined earlier through restriction endonuclease cleavage optimization experiments (see Before Starting 4., above). We used a 0.4 U/ µL FokI dilution in the present example, which typically provides near-complete (> 90%) DNA cleavage under our standard reaction conditions. Dilutions (1:9) were made with 1x PDB in a Terasaki plate microwell on ice and remained stable for at least 1 h before use.
?. [REPA execution]
Sample pre-equilibration for IISRE cleavage. Given that FokI is a mesothermic IISRE, samples need to be pre-equilibrated to its optimum reaction temperature prior to initiating cleavage. We found that a 5 min pre-equilibration at 37 °C is usually sufficient. To affect such, samples were transferred together from a 55 °C dry bath to a 37 °C dry bath. Note that pre-equilibration can occur for more extended periods (> 60 min) with no adverse consequences for SbtR binding. However, we routinely limit pre-equilibration to
?. [REPA execution]
IISRE cleavage. After pre-equilibration. IISRE cleavage was initiated by the addition of 1 µL 0.4 U/µL FokI to Tube #2 using a P2 micropipette. The sample was mixed thoroughly by finger flicking twice and snap shaking tube to consolidate liquid in tube bottom. The tube was returned to 37 °C dry bath block, and incubation allowed to resume for precisely 5 min. At that point, the cleavage reaction was stopped by the addition of 4 µL FokI loading buffer (FLB, 6x Orange Loading Dye with the addition of 1% SDS), mixed thoroughly as above, and storage at room temperature. With practice, it is possible to initiate IISRE cleavage for a new sample every 30 sec. Thus, with appropriate staging, one can have ten samples subjected to IISRE cleavage simultaneously. Table 2 illustrates the time points used in our SbtR REPA experiment.Table 2. SbtR REPA Timepoints  ABCD1  Sample    Experiment    Time (FokI)    Time (FLB)  2  1    Uncut DNA control    (n/a)  t = 4:303  2    FokI cleavage control    t = 0    5:00  4  3    200 nM SbtR    0:30    5:30  5  4    100 nM SbtR    1:00    6:00  6  5    50 nM SbtR    1:30    6:30  7  6    25 nM SbtR    2:00    7:00  8  7    13 nM SbtR    2:30    7:30  9  8    6.3 nM SbtR    3:00    8:00  10  9    3.1 nM SbtR    3:30    8:30  11  10    1.6 nM SbtR    4:00    9:00  Note that as Sample #1 is the uncut DNA control and does not require any FokI addition, FLB may be added at any time after the pre-equilibration step. We routinely choose the t = 4:30 timepoint as this provides a comparable time of exposure at 37 °C.
ABCD1  Sample    Experiment    Time (FokI)    Time (FLB)  2  1    Uncut DNA control    (n/a)  t = 4:303  2    FokI cleavage control    t = 0    5:00  4  3    200 nM SbtR    0:30    5:30  5  4    100 nM SbtR    1:00    6:00  6  5    50 nM SbtR    1:30    6:30  7  6    25 nM SbtR    2:00    7:00  8  7    13 nM SbtR    2:30    7:30  9  8    6.3 nM SbtR    3:00    8:00  10  9    3.1 nM SbtR    3:30    8:30  11  10    1.6 nM SbtR    4:00    9:00
?. [REPA analysis]
Densitometry. Labeled DNA products were quantitated using the DNA analysis module in LI-COR Image StudioTM 5.2 software. A detailed procedure may be found at https://dx.doi.org/10.17504/protocols.io.mcyc2xw. For two-label experiments such as REPA, we find that analyzing each channel independently is preferred. The densitometry of test and control DNAs from our SbtR REPA experiment is shown in Figure 6 with the data analysis in Table 4.
?. [REPA analysis]
Cleavage protection determination. Cleavage protection was determined using the following equation:% cleavage = X/(X + T)where X is the cleaved DNA for one probe (test or control), and T is its corresponding intact DNA. For our SbtR REPA experiment, the calculated % cleavage values as a function of SbtR concentration are shown in Table 4 and graphically in Figure 7 for both the test probe, which contains a consensus SbtR binding site and a control DNA. From this study, we find that the IC50 for FokI cleavage inhibition was 25 nM SbtR under these reaction conditions. This value compares favorably with those determined previously (https://dx.doi.org/10.1371/journal.pone.0159408).Table 4. REPA Results   ABCDEFGH1  Sample    Experiment    Test (T)*    Test (X)    Test (%X)    Control (T)    Control (X)    Control (%X)  2  1    Uncut DNA control    2490    2.36    0    9130    0    0  3  2    FokI cleavage control    133    1070    89    43.3    8040    99  4  3    200 nM SbtR    1720    15.7    1    29.9    7960    100  5  4    100 nM SbtR    1710    49.3    3    35.9    7710    100  6  5    50 nM SbtR    1370    393    22    54.5    8150    99  7  6    25 nM SbtR    751    793    51    47.1    7900    99  8  7    13 nM SbtR    461    1540    77    41.6    8630    100  9  8    6.3 nM SbtR    319    1250    80    67.1    7420    99  10  9    3.1 nM SbtR    219    1230    85    61.1    7010    99  11  10    1.6 nM SbtR    135    1240    90    44.1    7320    99
ABCDEFGH1  Sample    Experiment    Test (T)*    Test (X)    Test (%X)    Control (T)    Control (X)    Control (%X)  2  1    Uncut DNA control    2490    2.36    0    9130    0    0  3  2    FokI cleavage control    133    1070    89    43.3    8040    99  4  3    200 nM SbtR    1720    15.7    1    29.9    7960    100  5  4    100 nM SbtR    1710    49.3    3    35.9    7710    100  6  5    50 nM SbtR    1370    393    22    54.5    8150    99  7  6    25 nM SbtR    751    793    51    47.1    7900    99  8  7    13 nM SbtR    461    1540    77    41.6    8630    100  9  8    6.3 nM SbtR    319    1250    80    67.1    7420    99  10  9    3.1 nM SbtR    219    1230    85    61.1    7010    99  11  10    1.6 nM SbtR    135    1240    90    44.1    7320    99