Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Materials and Reagents

1. Pipette tips 
   
2. 15 ml conical tubes (USA scientific, catalog number: 1475-1611 )
   
3. Black 1.5 ml Eppendorf tubes (Argos Technologies, catalog number: T7456-001 )
   
4. 16 gauge needle (BD, catalog number: 305197 )
   
5. Fluorescein labeled AAVS1 sense DNA strand with the following sequence:
   5’-TGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCG-3’
6. AAVS1 anti-sense strand with the sequence: 5’-CGCCCAGCGAGCGAGCGA GCGCCGAGCCCCAACCGCCGCCA-3’
   Note: DNA can be synthesized using any synthesis facility such as IDT services at a 100 nanomole scale. The sense strand can be labeled at the 5’ end with fluorescein (6-FAM).
   
7. Purified recombinant AAV Rep68 protein was expressed in E.coli and purified using Ni-NTA affinity column followed by a gel filtration column as described previously (Musayev et al., 2015)
   
8. Sodium hydroxide (NaOH)
   
9. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
   
10. 2-Amino-2-(hydroxymethyl)propane-1,3-diol (Tris) (Sigma-Aldrich, catalog number: T1503 )
    
11. Ethylendiaminetetraacetic acid (EDTA) (Gold Bio, catalog number: E-210-1 )
    
12. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gold Bio, catalog number: H-401-500 )
    
13. Tris(2-carboxyethyl)phosphine (TCEP) (Gold Bio, catalog number: TECP )
    
14. Double distilled water
    
15. Q1 buffer (see Recipes)
    
16. Q2 buffer (see Recipes)
    
17. TES buffer (see Recipes)
    
18. Binding buffer (see Recipes)
    

Equipment

1. Pipettes 
   
2. MonoQ anion-exchange column (GE Healthcare, catalog number: 17-5166-01 )
   
3. HiTrap 5 ml desalting column (GE Healthcare, catalog number: 11-0003-29 )
   
4. GE Healthcare AKTA purifier
   
5. Labconco Freezone 2/5 Benchtop lyophilizer
   
6. Thermo Scientific NanoDrop ND-2000c spectrophotometer (Thermo Fisher Scientific, model: NanoDrop^TM 2000/2000c )
   
7. Denville IncuBlock heating block
   
8. ISS PC1 fluorimeter (ISS, model: PC1^TM )
   

Software

1. Microsoft Excel
   
2. GraphPad Prism 7^TM
   
3. Vinci Instrument control and data acquisition software from ISS
   

Procedure

1. Preparation of fluorescein labeled AAVS1 DNA
   1. Fluorescent labeled DNA can be purified by HPLC or using preparative acrylamide gel electrophoresis (PAGE) directly by the synthesis facility or can be purified by anion-exchange chromatography as described next. DNA is dissolved in 500 μl of Q1 buffer and injected into a MonoQ anion-exchange column. DNA was purified using a linear gradient from buffer Q1 (100 mM NaCl) to 100% Q2 buffer (1 M NaCl).
      
   2. Fractions are desalted using a 5 ml GE HiTrap desalting column pre-equilibrated with water. DNA elutes in the void volume.
      
   3. Collected DNA fractions are placed in a 15 ml conical tube, frozen and placed in a Labcono Freezone 2.5 lyophilizer overnight. To allow drying of the sample, the lid of the 15 ml conical tube should be pierced with a needle 4-5 times. The DNA should be fibrous and dry by the next day.
      
   4. After lyophilization, resuspend the DNA in 100 μl of TES buffer. Measure the DNA concentrations using a Thermo Scientific NanoDrop ND-2000c spectrophotometer using the calculated extinction coefficients of the DNA oligonucleotides at a wavelength of 260 nm.
      
   5. To prepare the double-stranded DNA, combine the two DNA strands in a ratio where the non-labeled strand is in 1.1 molar excess with respect to the fluorescein-labeled strand. Place the DNA strands in a black 1.5 ml microcentrifuge tube. Using a Denville IncuBlock heating block, heat the DNA at 99 °C for 3 min. Then, turn off the heating block and leave the DNA cooling to room temperature.
      
   6. The final DNA concentration is calculated using the number of moles of the labeled-DNA strand and the final volume after mixing the two strands.
      
      
2. DNA binding studies with Rep68
   Rep68 concentration is determined by absorbance at 280 nm using the extinction coefficient calculated from the Rep68 amino acid sequence using EXPASY Protparam site (http://web.expasy.org/protparam/). Prepare different concentrations of Rep68 in binding buffer and mix with DNA allowing the binding reaction to reach equilibrium at 20 °C for 20-30 min. For other proteins, a preliminary experiment needs to be performed to obtain a rough estimate of the concentrations to be used. Titrations are carried out using a final 5 nM DNA concentration in binding buffer. A typical binding experiment is performed as follows:  
   
   1. It is recommended that the PC-1 fluorimeter is turned on the night before the experiment to stabilize the instrument (Figure 1). Start the Vinci software on the computer. Access instrument control and set the excitation and emission filters to 492 nm and 528 nm, respectively.
      
   2. Turn-on the lamp of the PC1 fluorimeter and let it warm up for at least 1 h.
      
   3. Calculate the amount of Rep68 stock solution needed to achieve the following final concentrations in the binding reactions: 0, 10, 25, 50, 75, 100, 150, 200, 300, 400 and 500 nM.
      Generally, three replicates are done for each concentration.
      
   4. Mix the different Rep68 concentrations with 5 nM DNA in a final volume of 300 μl. Incubate samples for 20-30 min away from light.
      
   5. Measure the anisotropy value of each concentration point using the ‘Single point Polarization’ function in the Vinci software and record 10 measurements per concentration.
      
      
      Figure 1. PC1 fluorimeter from ISS
      

Data analysis

1. Open all the data in Microsoft Excel and for each concentration calculate the average anisotropy value from the 10 measurements.
   
2. Make a new Graphpad project using the Rep68 concentration as the x values and the average anisotropy as the y value.
   
3. Subtract the DNA anisotropy value from all concentrations.
   
4. Using Prism 7^TM (GraphPad), perform a nonlinear fitting using a single specific binding model. Prism uses the following equation:
   
   
   Where,
   A_max is the maximum anisotropy value at saturation,
   C_x is the concentration of Rep68,
   K_d is binding constant.
   
5. To calculate the fraction of DNA bound, the maximum value obtained from the fitting is used as the anisotropy value at saturation when all DNA is bound (A_max). The fraction of DNA bound at each concentration is the anisotropy at each concentration (A_x) divided by Amax. This is shown in Figure 2A. Anisotropy values (A_x) at each concentration (Group A) were converted to fraction bound (Group B).
   
6. Below is an example of a typical experiment.
   
   
   Figure 2. Data analysis. A. Anisotropy data and correction for fraction bound; B. Nonlinear Fitting of the data to specific binding model.
   

Notes

1. It is recommended that during the incubation step, samples be kept away from light and incubated at the temperature of the instrument by means of a water bath or heating/cooling block.
   
2. Protein stocks are stored at high concentration (~0.1-0.5 mM) at -80 °C and are diluted in binding buffer to the required concentrations. Generally, a series of dilutions from 100 μM to 100 nM are prepared to cover the concentration range of the titration curve.
   
3. If incubation time is not known, this can be determined by incubating sample and reading anisotropy every five minutes. Equilibrium is estimated when the measured anisotropy remains constant.
   

Recipes

1. Q1 buffer
   10 mM NaOH
   100 mM NaCl (pH 12.0)
   
2. Q2 buffer
   10 mM NaOH
   1 M NaCl (pH 12.0)
   
3. TES buffer
   10 mM Tris
   100 mM NaCl
   1 mM EDTA (pH 8.0)
   
4. Binding buffer
   25 mM HEPES
   200 mM NaCl
   1 mM TCEP (pH 7.0)
   

Acknowledgments

This work was supported by NIH grant R01-GM092854. The authors declare no conflict of interests.

References

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2. Hickman, A. B., Ronning, D. R., Perez, Z. N., Kotin, R. M. and Dyda, F. (2004). The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces. Mol Cell 13(3): 403-414.
   
3. Im, D. S. and Muzyczka, N. (1990). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61(3): 447-457.
   
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7. Musayev, F. N., Zarate-Perez, F., Bishop, C., Burgner, J. W., 2nd and Escalante, C. R. (2015). Structural insights into the assembly of the adeno-associated virus type 2 Rep68 protein on the integration site AAVS1. J Biol Chem 290(46): 27487-27499.
   
8. Weitzman, M. D., Kyostio, S. R., Kotin, R. M. and Owens, R. A. (1994). Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci U S A 91(13): 5808-5812.
   
9. Wonderling, R. S., Kyostio, S. R. and Owens, R. A. (1995). A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities. J Virol 69(6): 3542-3548.
   
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