DNA Condensation Using Self-Assembled Peptides

DNA Condensation Using Self-Assembled Peptides Angela m. Jimenez, Class of 2008, Major: Chemical Engineering Mentor: Raymond S. Tu, Department of Chemical Engineering ABSTRACT Counterion-mediated DNA-condensation is critical to most cellular DNA activities that take place in the cell, from chromosome packaging to control over translational mechanisms. Creating synthetic systems to manipulate DNAcondensation may be useful for the development of biotechnologies for gene encapsulation and DNA-separation processes. Our approach is to use peptide based building blocks to study the effect of dynamic assembly on the DNA condensation process. The peptide used in this investigation has α-helix secondary structure and contains 23 amino acids. The peptide has an amphiphilic nature with hydrophobic residues on one side and hydrophilic residues on other side. The dynamic nature of this molecule can be examined by the study of its response to polyelectrolytes (DNA) in monovalent salts (NaCl) at various concentrations. This response permits the characterization of the ensemble average α-helix structure with changing counterion concentration. Our studies have shown that the secondary α-helix structure of the peptide can be manipulated as a function of the concentration of salt. Moreover, we show that DNA complexes with the alpha helical peptide solution, resulting in self-assemblies that are characterized for surface charge and aggregate size. Future studies will explore the design of more peptides with various spatial distribution of charge to understand the effect of charge spacing on the DNA condensation and assembly process. KEYWORDS: Counterion-mediated DNA-condensation; peptides; secondary structure; self-assembly; transfection INTRODUCTION DNA delivery systems have been classified as viral-mediated systems and as non viral-mediated systems. The viral-mediated systems have proven to be very effective in the DNA delivery, reaching high efficiencies in both delivery and expression. Although these viral systems are very efficient in the DNA transfection, some of the limitations that they carry along are toxicity, restriction on targeting of specific cells, production and packaging problems, limitation on DNA-carrying capacity, and high cost. For these reasons, the investigation of non-viral systems has become a very desirable and important task[1]. Several non-viral techniques to facilitate the transport of DNA into cells that involve the construction of synthetic vectors are currently being studied. These synthetic vectors are typically constructed by binding the negatively charged DNA with synthetic biomolecules, such as positively charged peptides or cationic lipids, to generate DNA-complexes. The resulting synthetic complexes can dock with the cell surface by binding to the cell membrane. This binding is thought to be initiated by the affinity between the net charge of the vectors and the cells; if the vectors have an overall positive charge, they would bind nonspecifically to the negatively charged cell membrane[2]. The ability to thus deliver genes to specific cells is highly desired, as it would reduce toxicity, side effects, and dosage requirements. Therefore, considerable effort has been invested to develop synthetic vectors capable of efficiently targeting gene delivery to specific cells[3]. The efficiency of DNA transfection depends on the DNA delivery (i.e., fraction of DNA molecules that get into the nucleus) and the DNA expression (i.e., the fraction of nuclear DNA molecules that undergo transcription). The process of DNA transfection can be summarized in three general steps: (1) DNA complexation and condensation, (2) endocytosis and (3) nuclear targeting. In this multi-step process, even when the DNA complexes are transported to the nucleus from outside the cell, they are typically degraded within the cell. Therefore, identifying and overcoming the difficulties in each step of the DNA journey to the nucleus can improve delivery, as well as the efficiency of transfection in general[1]. 24 Journal of Student Research

Angela Jimenez is originally from Co- lombia. When she was in high school, she greatly enjoyed taking math and science courses which motivated her to study Chemical Engineering at The City College of New York. After taking a class with Professor Raymond Tu during her junior year, she saw the opportunity to be exposed to the world of research and gain valuable experience and skills in the field of engineering. She persuaded him to give her the opportunity to conduct research and joined his group in June 2006. After working in the lab for a few months, she found out about the National Institutes of Health scholarship that is provided to minority students who conduct research and are interested in pursuing a Biomedical Engineering concentration. She applied for the scholarship as she was conducting research on biomolecules exposed to other research areas; one such instance is the research and now receives financial support to continue with the research experience that she had last summer at Harvard University, where project that investigates DNA condensation using self assembled she examined the interaction of cationic and anionic surfactants. peptides. Through that opportunity, Angela was able to secure an internship This work is carried out in collaboration with Vikas P. Jain, a doc- in France to conduct research for six months. After her graduation toral student under Professor Tu, with his guidance and constant en- from CCNY in May she will travel to France, and later this year she couragement. This opportunity has not only helped Angela to un- will be applying to graduate school in the hope of pursuing a Ph.D. derstand the value of research but also to make connections and be in Chemical and Biomolecular Engineering. Professor Raymond Tu focuses his research on search has two key aspects. First, his group designs and synthesizes designing synthetic peptides that self-assemble to yield both bio- a peptide with a particular amphiphilic molecular architecture. Sec- logically specific binding and useful materials properties. Dr. Tu s re- ond, his group characterizes the peptides for their self-assembled structure and their ability to mimic biological binding selectivity. The applications of this bottom-up peptide design include targeted drug delivery, biosensing, synthetic biomimetic materials, and separation processes. Dr. Tu came to The City College in 2005, after receiving his Ph.D. from The University of California, Santa Barbara. He praises the Grove School of Engineering for the collegiality of its faculty and the dedication of its students. He continues to empower his students to use whatever tools are available to pursue their goals, both in the classroom and in the lab. Dr. Tu emphasizes the importance of student research. The experimental problems presented in the lab differ fundamentally from classroom problems. Experimental problems encountered in student research have a vast number of parameters to explore, where intrepid ideas and keeping organized are keys to success. V o l u m e 1, M ay 2 0 0 8 25

Our work aims to develop a systematic approach to design peptides for DNA condensation/complexation. The key challenge is to create a model peptide-based DNA sequence that provides a basis for the study of DNA complex formation. This work proposes to apply a non-viral vector, cationic peptides, to encapsulate DNA. This technique can be an efficient agent for transfection of eukaryotic cells because the peptide-dna complex (condensed state) protects the DNA from nucleases, preventing degradation, and allows the complex to pass more easily through cellular membrane openings[4]. The challenge is to promote a system that is highly efficient in DNA delivery/ expression[1]. EXPERIMENTS AND RESULTS In order to address the difficulties in the process of DNA delivery, we are concentrating this study on the phenomenon of DNA complexation and condensation. Establishing control over this first stage in the process of DNA transfection will improve the journey of the DNA complex to the nucleus. To condense DNA from an extended linear phase into a compact phase capable of transfection requires an extraordinary degree of hierarchical packaging. Some of the energetic barriers that are involved in this condensation process involve the loss of configurational entropy of the long DNA molecule, tight bending of the stiff double helix and the electrostatic repulsion of the negatively charged DNA phosphates[5]. This research studies DNA condensation using rationally designed peptides constructed to have two key behaviors: (1) transient α-helix secondary structure and (2) amphiphilicity. Peptides are short polymers made from amino acids; they can be fabricated synthetically in the laboratory, employing solid phase synthesis. One can precisely control the spatial, physical and chemical properties of peptides by controlling the sequence of amino acids. Rationally designed peptides with a well defined secondary structure can self-assemble and distribute chemical functionality (charge) in a controlled fashion, offering a versatile tool to study DNA condensation process as a function of peptide sequence. There are three components described in this study: (1) peptide design, (2) structural characterization with circular dichroism spectroscopy, and (3) characterization of the DNA condensation behavior with particle size analysis and zeta potential measurements. Peptide Design We applied the following rationale to select the sequence shown in Figure 1. Counterion interactions have been shown theoretically and experimentally to be the driving force for DNA packing[5]. Research suggests that to have attractive forces between DNA and the peptide, the peptide s surface charge should be +3 or greater. In order to design our peptide sequences, we are taking this +3 charge as a rule of thumb. This Figure 1: Rationally designed peptide sequence for DNA condensation experiments. The peptide is comprised of six amino acids shown at bottom. 26 Journal of Student Research

condition is sufficient, as the goal of this project is to study the non-specific interaction between the peptide and DNA. Future sequences will include specific interactions between the peptide and particular DNA sequences. To control self-assembly we need the peptide to be amphiphilic. Amphiphilicity means that one face of the helix is hydrophobic and the other face is hydrophilic. To accomplish this we design the peptide using an algorithm that combines (1) intrinsic folding propensities and (2) helical periodicity. This experiment employed a peptide that contained six different types of amino acids. These amino acids were organized to design a peptide with the correct periodicity of non-polar and polar amino acids to form an amphiphilic α-helix structure. These particular amino acids were chosen for the following reasons: Leucine (Leu) and alanine (Ala) have hydrophobic propensities and are strong α-helix formers[7]. Tryptophan (Trp) is a hydrophobic α-helix former amino acid and allows us to quantify the concentration of peptide that has been deposited on the substrate with UV/VIS absorption[7]. Glutamic acid (Glu) is a negatively charged hydrophilic amino acid with a strong helix propensity[7]. Lysine (Lys) is a positively charged hydrophilic amino acid that promotes the binding of DNA[6,7]. Histidine (His) is a strong α-helix former that is placed in the N-terminal of the sequence[7]. Structural characterization with circular dichroism Circular Dichroism (CD) spectroscopy is used to measure the ensemble average secondary structure of the peptide. In our case, we are characterizing the fraction of the peptide that is α-helical at various salt concentrations and in the presence of DNA. This measurement is accomplished by passing circularly polarized light through the solution that contains an optically active molecule, our peptide. The instrument measures the difference in absorption between the left circularly polarized light and the right circularly polarized light. Due to a structural asymmetry of the optically active component in the solution, the absorption of polarized light varies as a function of wavelength. Secondary structure is determined by CD spectroscopy in the far-uv region (190-250nm). At this wavelength, the peptide bond is the chromophore. The absence of chiral molecules results in zero CD intensity, while an ordered chiral structure results in a characteristic spectrum for each type of secondary structure. This method determines the ensemble average distribution of α-helices, β-sheets and random coils in the path of the light. The basic standard conformations of known polypeptides is illustrated in Figure 2. To fit the secondary structure, the method of least squares is applied[8]. Figure 2: Characterisitic CD spectrum where blue is α-helix conformation and red is random coil. A peptide solution is prepared by dissolving 2mg of peptide into 4ml of deionized water. Also, a 2 molar (2M) NaCl solution is made dissolving 0.7013 g of NaCl in 6 ml of deionized water. Then solutions of different concentrations are made by the combination of these two solutions as needed. The CD spectra of these solutions are taken in order to evaluate the degree of peptide α-helicity as a function of salt concentration. We observe that as the salt concentration is increased, the peptide α-helical conformation is increased. At 0M NaCl, the major contribution to the CD spectrum is mostly from random coils. The molar ellipticity for α-helices exhibits a minimum in CD at 222 nm. Figure 3 illustrates that as salt concentration increases, molar ellipticity decreases at 222 nm. This graph can be used to calculate the equilibrium constant of the reversible reaction between unfolded and folded peptide. This constant is calculated by drawing a smooth curve passing through the maximum number of points, then making two lines parallel to the x-axis. One of the lines is drawn through the y-value where x=0 and the other one is drawn tangent to the curve. Tie lines are drawn at particular NaCl concentrations and the fraction of the line above the curve represents the folded peptide and the fraction of the same line below the curve represents the unfolded peptide. Volume 1, May 2008 27

Figure 3: Molar ellipticity at 222nm as a function of salt concentration. The ratio of the fraction of folded peptide to unfolded peptide is the equilibrium concentration. For example at 250 mm NaCl we have approximately k folded /k unfolded = 4.1. Experiments with DNA Experiments are performed using DNA to examine the condensation/complexation phenomenon by the peptide. One of the visual observations while doing this experiment is the precipitation of the sample and, therefore, a decrease in the CD intensity as the aggregate begins to scatter both the right and left circularly-polarized light equally. Additionally, zeta potential changes were measured to quantify the point of charge inversion. CD of DNA-peptide An aliquot quantity of 1g/l solution of DNA was added to a 1g/l peptide solution, and the solution was properly mixed. Both the peptide and the DNA solutions were prepared with 50 mm NaCl solution. At this concentration of NaCl, the peptide has a folded fraction of approximately k folded /k unfolded = 0.13. After every addition of DNA to the peptide solution CD measurements were done, as the DNA concentration increased there was a decrease in the absorption intensity. Wavelength (nm) Figure 4: Circular dichroism for the peptide solution as it changes in conformation. Each curve represents the CD of the peptide at different salt concentrations. 28 Journal of Student Research

Zeta Potential (mv) 50 40 30 20 10 0-10 -20-30 -40 Figure 5: -50 0 10 20 30 40 50 60 70 80 90 100 concentration. DNA (mole %) Zeta potential of the DNA-peptide complex as a function of DNA Zeta potential measurement as a function of DNA To quantify the charge associated with the aggregated systems, zeta potential measurements are done as a function of DNA concentration. Zeta potential is a measure of the electric potential at the double layer of the aggregated structure. An aliquot of DNA solution at 1 mg/ml is titrated to a peptide solution of 1 mg/ml. After each titration step, zeta potential measurements were taken as illustrated in Figure 5. At low DNA concentration, (mostly peptide in solution) the zeta potential is positive. As the DNA concentration is increased, the zeta potential sharply transitions to negative at 70% (mol) DNA. DISCUSSION We presume that the random coil formation in DI water is a result of the overall +3 positive charge of the sequence, where intramolecular repulsion due to the electrostatic repulsion prevents the formation of helices. Addition of salt to this solution screens the charge-to-charge interaction, increasing the ensemble average percentage of α-helices. In summary the peptide has a random structure in DI water and forms an α-helix conformation in high concentrations of salt solution, as seen in Figure 4. We concluded that the DNA concentration increases with a decrease in absorption intensity because a complex forms, causing a precipitation reaction. This can be explained in the following way: (i) the peptide is folded into an α-helix as it binds to the DNA, (ii) the helical peptide assembled at the DNA surface creates a higher interfacial energy, and (iii) the peptide: DNA complexes self-assemble to reduce the solution free energy. One clear observation from this experiment is that the peptide is capable of condensing DNA. We interpret the increase of the zeta potential with increasing DNA concentration and its shift to negative at 70% (mol) as the likelyhood that there is a critical DNA concentration that promotes the peptides to transition to α-helices. As this happens, the peptide-dna complexes aggregate together and fall out of solution. These aggregated or condensed structures may be used to explore the transfection of cells in future studies. CONCLUSIONS We believe that our rationally designed peptide is, indeed, capable of folding into α-helices and condensing with DNA. The structural aspect of this phenomenon is verified with circular dichroism, and the condensation behavior is examined with zeta potential measurements. Future work with this peptide aims to evaluate the details of the nanostructure of the peptide DNA complex. We plan to try different approaches, such as Cryo-electron microscopy and small angle neutron/x-ray scattering, to analyze the complex formation. Likewise, we will design more peptides with the same composition but with different charge distributions to study the effect of charge spacing in the DNA condensation process. To analyze specific binding, we plan to include amino acid sequences that mimic the contacts made by a class of DNAbinding proteins known as transcription factors. ACKNOWLEDGMENTS This research was funded by the National Institutes of Health. REFERENCES [1] D. Luo, W.M. Saltzman, Synthetic DNA delivery systems, J. Nat. Biotechnol. 18 (2000) 33-37. [2] J. N. Israelachvili, Controlled release systems for DNA delivery, Intermolecular and Surface Forces: With Applications to Colloidal and Biological Systems; Academic Press, London, 1985. [3] A. K. Pannier, L. D. Shea, Controlled release systems for DNA delivery, J. Mol. Ther. 10 (2004) 19-26. [4] V.A. Bloomfield, J. Biopolymers. 6 (1997) 334-341. [5] V.A. Bloomfield, DNA condensation by multivalent cations, J. Biopolymers. 44 (1997) 269-282. [6] H. Lodish, A. Berk, L. S. Zipursky, P. Matsudaria, D. Baltimore, J. Darnell, Molecular cell biology, New York, New York, 1995. [7] P. Y. Chou, D. Y. Fasman, Prediction of protein conformation, J Biochemistry. 13 (1974) 222-245. [8] S. Brahms, J. Brahms, Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism, J. Mol. Biol. 138 (1980) 149-178. Volume 1, May 2008 29