1. Figure 5 Sample mobile shift assay gel. The 3 rd lane from the left shows a significant “shift” in the protein location, representing the proteins which were bound to the target complex. Figure 3 An example of a DNA sequence alignment, similar to the one we used to determined the validity of ZFN sequences once ligated into host E. coli cells. A Comparative Study of Zinc Finger Nuclease Activity Eric Hall, Laura Young, Ronnie Winfrey & Dan Voytas RET Summer Internship 2007 Abstract In the ever advancing field of gene therapy, one challenge faced is the targeting of specific DNA sequences for modification. One promising response is the development of engineered zinc finger nucleases. Zinc finger nucleases are a tool for targeting DNA in plants, animals, and insects. Current research in this novel area of study has been focused towards improving the ability to engineer modular assembly zinc finger arrays. One obstacle is the varying binding affinity among different zinc finger arrays. Our research involved comparing the effect of replacing the third finger on a known poor-binding zinc finger array. Over the course of our time in the lab we constructed the zinc finger nucleases and our preliminary data indicates that we successfully inserted the nucleases into the vector. At this time, we are awaiting materials to complete the gel shift which will reveal the binding affinity of our various nucleases. Background A zinc finger array is a modular assembly of three “fingers” of DNA each of which code for a protein that binds to a specific three base pair sequence on target DNA. The finger array is bound to a zinc ion which in turn can be fused to a restriction enzyme. When used in conjunction with a second zinc finger/restriction enzyme, the dimer formed can be used to pinpoint a precise location for cutting double-stranded DNA. It is this ability to precisely open a location on DNA that holds promise in the field of gene therapy. With this technology, organisms such as plants can be modified to be more disease resistant or have higher yields. In humans, this technology can improve the success of gene therapy in the treatment of diseases such as cystic fibrosis or Parkinson’s. A zinc finger array is a modular assembly of three “fingers” of DNA each of which code for a protein that binds to a specific three base pair sequence on target DNA. The finger array is bound to a zinc ion which in turn can be fused to a restriction enzyme. When used in conjunction with a second zinc finger/restriction enzyme, the dimer formed can be used to pinpoint a precise location for cutting double-stranded DNA. It is this ability to precisely open a location on DNA that holds promise in the field of gene therapy. With this technology, organisms such as plants can be modified to be more disease resistant or have higher yields. In humans, this technology can improve the success of gene therapy in the treatment of diseases such as cystic fibrosis or Parkinson’s. Previous research has been conducted by the Voytas lab regarding the binding affinity of various G series three finger arrays (G referring to the first nucleotide of the target DNA). From that research it was indicated that there was a correlation between binding success and the type of third finger. For our project, we set out to construct fingers in order to compare the effect of replacing the poor-binding third finger with a more positive finger. Previous research has been conducted by the Voytas lab regarding the binding affinity of various G series three finger arrays (G referring to the first nucleotide of the target DNA). From that research it was indicated that there was a correlation between binding success and the type of third finger. For our project, we set out to construct fingers in order to compare the effect of replacing the poor-binding third finger with a more positive finger. Research Question How does the replacement of one finger on a known, poor- binding three finger zinc finger nuclease affect binding affinity? How does the replacement of one finger on a known, poor- binding three finger zinc finger nuclease affect binding affinity? Methods Our research required two general processes: 1) assembling the 3- finger ZFN arrays and 2) determining their relative effectiveness. Assembly of the ZFNs happens through a series of restriction enzyme digests and transformations into competent E. coli cells. Each “finger” is obtained from a library of frozen plasmids. These plasmids consist of a 3-4kb backbone with the ~100 base pair ZFN sequence. To assemble a 3-finger ZFN array, one follows this basic protocol (Figure 1): 1.Open up the F1 vector plasmid with AgeI and BamHI restriction enzymes. 2.“Cut out” the F2 finger sequence fragment using Xma and BamHI. 3.Ligate the F2 fragment into the F1 vector using T4 DNA ligase. 4.Repeat steps #1-3 using the “new” F1+2 vector and the F3 plasmid. 5.After each ligation, our plasmid was transformed into H10B E. coli cells and grown at 37°C overnight on LB+Amp media (Figure 2). Next, we wanted to determine the affinity and specificity of our newly constructed 3-finger arrays. We had two choices – a gel shift assay or a bacterial 2-hybrid assay. Because of time constraints we opted for the simpler gel shift assay. Preparation for the gel shift began with the ligation of our 3-finger fragment into the gel shift vector, E. coli H10B. This was done using the same electroporation technique as in previous experiments. These cells were exposed to isopropyl-beta-D-thiogalactopyranoside (IPTG), a synthetic analog of lactose which induces protein production using the lac operon. Once translation of the plasmid had begun, concentrations of our zinc finger protein increased. These proteins were then exposed to the target DNA sequence in the form of “hairpin” oligos and run on an agarose gel. In this situation, if the ZFNs bind to the target oligos, these larger complexes will run slower on the gel and be “shifted” when compared to the non-bound oligos. Data Obtained During the course of our experiment, many types of data were collected and analyzed. Frequent analysis of data allowed us to determine whether or not we should proceed to the next step. Here is a sample of the types of information we used… DNA sequence alignment of our ZF array fragments showed 100.0% matching between our constructed ZFN plasmids and our desired sequence (Figure 3). The successful growth of our transformed E. Coli cells demonstrated adequate expression of our desired sequence (Figure 2). The presence of a 100-300bp band on our 0.8% agarose gel after electrophoresis is indicative of the successful insertion of our ZFN fragment into our vector plasmid (Figure 4). Our gel shift assay would have shown us whether or nor the ZFN protein bound to our target oligo (DNA sequence). Because of time restraints, this assay was not completed. Figure 5 shows a sample mobile shift assay gel. Discussion of Results Our preliminary data showed good quality ligations of our ZFN into vector plasmids. The presence of 100-300bp bands on our 0.8% agarose gels represents our ligated ZFN fragment, and these bands were present after each ligation. Sequencing data also showed a high quality ligation into our vector plasmids. Due to time restraints, our gel shift assay was not completed, but would have shown – in a qualitative sense – whether or not our ZFN protein bound to its target DNA sequence. References David Wright, et. al. Standardized Reagents and Protocols for Engineering Zinc Finger Nucleases by Modular Assembly. Nature Protocols. Volume 1 No.3. 1637-1652. Acknowledgements We would like to thank Ron Winfrey for his guidance and support during our research this summer. In addition, we would like to thank Dan Voytas for opening his lab to us. We also appreciate the help and patience of Jeff, Pete and Fengli as we made our way through the summer. Finally, thank you to the National Science Foundation for making this internship possible for all of us this summer. Figure 1 “Standardized Reagents and Protocols for Engineering Zinc Finger Nucleases by Modular Assembly”, Nature Protocols, Vol. 1 No. 3, 2006 AgeI/BamHI digest Isolate vector backbone XmaI/BamHI digest Isolate F2-encoding fragment AgeI/BamHI digest Isolate vector backbone XmaI/BamHI digest Isolate F3-encoding fragment Ligate Figure 2 0.8% agarose gel showing distinct 200-300bp fragments, indicating the successful ligation of our ZFN sequences into the vector plasmids. Figure 4 E. Coli H10B after ~15hrs growth at 37°C. Visible colonies are those which have taken up the desired ZFN sequence and can “survive” on media infused with ampicillin.