Browsing by Subject "DNA"

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Bridging the gap between theory, experiments and simulations of nanochannel confined DNA
(2020-08) Bhandari, Aditya Bikram
The study of nanochannel confined DNA has garnered substantial attention since the early 2000's owing to its application in genome mapping, the coarse-grained counterpart to DNA sequencing, which is an indispensable tool in biological research. However, our understanding of the physics behind confined DNA is rather simplified and incomplete. Thus, theory, simulation and experiment have by and large been at odds with one another. The results of this dissertation are aimed at understanding and attempting to resolve the source of these discrepancies. Our strategy for this dissertation is three-pronged. First, we revisit a historically cited explanation for the discrepancies - the lack of understanding behind the wall depletion length denoting the wall-DNA electrostatic interactions. Second, we considered the intersection of theory and simulation, which recent developments have managed to bring sufficiently into accord. We found that the deviations between the fractional extension distributions predicted by an asymptotic theory and those observed experimentally, are not due to a breakdown of the theory, even for experimental conditions which typically do not strictly satisfy the asymptotic limits of the theory. This motivated a closer inspection of the theories to determine a missing link between theory and experiment. Finally, by studying a recently generated dataset of fractional extensions spanning a wide range of the experimental parameter space, we were able to isolate this missing link as the effect of long-range electrostatics in the system which are typically ignored in the simplified theories, wherein the DNA is assumed as a neutral polymer confined in a channel of a reduced effective channel size. We believe that our findings within this dissertation will provide a better understanding of confined polymers and, in particular, the nanochannel confined DNA system used in genome mapping, as well as provide new directions of study in the future.
Cataloging Essential Genes for Natural Transformation and Expanding Genetic Tools for Study of Archaea
(2023-04) Fonseca, Dallas
Horizontal gene transfer is a near ubiquitous way for organisms to acquire new geneticinformation. One mechanism of horizontal gene transfer is natural transformation, the uptake/incorporation of DNA from the environment into the genome. In Bacteria, DNA uptake has been well documented in numerous phylogenetically diverse species. Across all of these organisms, four operational steps have to occur. First, the pathway must be induced, entering cells into a state known as competence. Extracellular appendages on the cell then reach out from the cell surface and adsorb DNA. DNA is subsequently translocated through a transporter ComEC and bound by intracellular single-strand binding proteins for integration into the genome. In Archaea, no mechanism of natural transformation has been previously identifiedand homologs of bacterial competence genes are sparse. Presented here is the identification of the process of natural transformation in two distinct members of the Archaea. Using a variety of genetic techniques, I identify several components that are essential to the natural transformation pathway. This includes Type IV-like pili, putative membrane-bound substrate transporters, proteins predicted to bind DNA, as well as several hypothetical proteins. While this thesis provides the first catalog of genes essential to natural transformation, the exact mechanism that underlies this process is still elusive. The later chapters of this thesis will discuss preliminary approaches to determine the mechanisms of natural transformation mediated DNA uptake.
Chimeric Criminals
(2013-02-12) Kaye, David H.
According to some commentators, an obscure genetic condition known as chimerism “could undermine the very basis of the forensic DNA system” and force a reconsideration of “the entire project of forensic DNA.” This conclusion is as unfounded as it is unnerving. Chimerism is a consideration in, but not a real obstacle to DNA identification. This essay explains why.
Complexation Between DNA and Hydrophilic-Cationic Diblock Copolymers
(2017-08) Jung, Seyoung
DNA-polycation complexes (polyplexes) are gene delivery vehicles that offer a number of advantages over viral vehicles: high genetic payload capacity, low toxicity, and low cost. Among the many polycation architectures, hydrophilic-cationic diblock copolymers are particularly promising due to their versatility. Compared to the emphasis on biological evaluation of polyplexes, however, in-depth understanding of the polyplex formation process (and the parameters involved) is rather sparse. Thus, this thesis discusses the physics of polyplex formation. First, a collection of solution-state techniques are employed to elucidate the effects of diblock composition, ionic strength, DNA morphology, and pH on DNA-diblock binding. Specifically, the binding thermodynamics and strength - measured using isothermal calorimetry and circular dichroism spectroscopy, respectively - turn out to show a strong dependence on the cationic content in the diblock, leading to wide ranges of polyplex size, dispersity, and stability. Second, the effects of hydrophilic block structure, charge density, and polyplex formation route are investigated. It is found that bulkier hydrophilic blocks translate to larger polyplexes when DNA is in excess and that hydrophilic block solubility controls polyplex stability when the diblock is in excess. With fixed material and concentration, different polyplex sizes and dispersities can be achieved by varying solution ionic strength or DNA-polycation mixing route. To this end, the analysis of polyplex formation presented in this thesis provides original insights into physical variables that can be adjusted to tune the DNA-polycation binding behavior and resulting polyplex properties, which can aid in the guided development of polymeric DNA delivery systems.
Complexation of DNA with Polycationic Micelles
(2018-08) Jiang, Yaming
Interpolyelectrolyte complexation is a ubiquitous phenomenon that plays vital roles in biological systems and in design of responsive materials. However, precise control of polyelectrolyte complexes (PEC) has been challenging, particular for DNA-polycation complexes designed for gene delivery applications. Incorporating polyionic micelles is a promising strategy to tune PEC properties, but has been under-utilized in designing polymeric gene delivery vehicles. Herein, cationic micelles self-assembled from amphiphilic block polymers are complexed with double stranded DNA. The structure, composition, and stability of the resulting "micelleplexes" are characterized to probe the fundamental physics that govern the formation and properties of micelleplexes. With cationic AB+ micelles, complexation of linear semiflexible DNA and flexible poly(styrenesulfonate) were compared and the influence of polyanion chain flexibility was extracted and discussed. DNA length was found to strongly influence the size, composition, and colloidal stability of micelleplexes, whereas DNA topology (linear or circular supercoiled) has minimal influence. To improve the colloidal stability and reduce the size of micelleplexes that are composed of multiple micelles connected by bridging DNA chains, AB+C micelles with hydrophilic nonionic outer coronas of varying length were designed. The addition of the outer nonionic corona dramatically improves the colloidal stability of micelleplexes over a much wider charge ratio, and the outer corona length closely correlates to micelleplex size, zeta potential, and the average number of micelles per micelleplex. In addition, AB+C micelleplexes adopt a beads-on-a-string structure that resembles the organization of DNA in chromatin. Lastly, structure, composition, and stability of micelleplexes were closely compared with those of another typically studied family of DNA complexes, "polyplexes", which form between DNA and cationic homopolymers or AB+ diblock copolymers with a hydrophilic nonionic A block. Compared to the polyplexes, micelleplexes showed more than a 4-fold increase in gene transfection efficiency, which was attributed to the high positive charge content of micelleplexes.
Crystallization study of the resistance to cobalt and nickel repressor (RcnR) protein in complex with double-strand DNA
(2019-12) Li, Chao
This thesis included my work on the crystallization, data analysis and phasing attempts regarding RcnR in complex with a ds-DNA molecule .RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. We crystallized Escherichia coli RcnR with DNA that encompasses the DNA binding site. X-ray diffraction data were collected to 2.9 Å. The crystal belongs to space group P61/522, with unit cell parameters a = b = 73.65 Å, c= 153.77Å, α=β=90°, γ = 120°. The second and third part included my work on MauG and PqqB projects.
Data and code supporting: Simulations corroborate telegraph model predictions for the extension distributions of nanochannel confined DNA
(2019-08-12) Bhandari, Aditya Bikram; Dorfman, Kevin D; dorfman@umn.edu; Dorfman, Kevin D; Dorfman
Hairpins in the conformation of DNA confined in nanochannels close to their persistence length cause the distribution of their fractional extensions to be heavily left skewed. A recent theory rationalizes these skewed distributions using a correlated telegraph process, which can be solved exactly in the asymptotic limit of small but frequent hairpin formation. Pruned-enriched Rosenbluth method simulations of the fractional extension distribution for a channel-confined wormlike chain confirm the predictions of the telegraph model. Remarkably, the asymptotic result of the telegraph model remains robust well outside the asymptotic limit. As a result, the approximations in the theory required to map it to the polymer model and solve it in the asymptotic limit are not the source of discrepancies between the predictions of the telegraph model and experimental distributions of the extensions of DNA during genome mapping. The agreement between theory and simulations motivates future work to determine the source of the remaining discrepancies between the predictions of the telegraph model and experimental distributions of the extensions of DNA in nanochannels used for genome mapping.
Data for: Diffusion of Knots along DNA Confined in Nanochannels
(2020-08-05) Ma, Zixue; Dorfman, Kevin D; ma000052@umn.edu; Ma, Zixue; University of Minnesota Dorfman Research Lab
We study the diffusion of knots along relaxed DNA in nanochanels using a nanofluidic "knot factory" device for knot generation. The knot diffusion data for the article is published as "Diffusion of knots along DNA Confined in Nanochannels" in Macromolecules. The data includes DNA images before and after knot generation and all the data used to generate the figures in the article.
Data for: Diffusion of knotted DNA molecules in nanochannels in the extended de Gennes regime
(2021-04-19) Ma, Zixue; Dorfman, Kevin D; ma000052@umn.edu; Ma, Zixue; University of Minnesota Dorfman Research Lab
We study the effect of knots on DNA diffusion in nanochannels using a nanofluidic "knot factory" device for knot generation. The unknotted and knotted DNA diffusion data for the article is published as "Diffusion of knotted DNA molecules in nanochannels in the extended de Gennes regime" in Macromolecules. The data includes DNA images before and after knot generation and all the data used to generate the figures in the article.
Data for: Extension distribution for DNA confined in a nanochannel near the Odijk regime
(2019-09-19) Chuang, Hui-Min; Reifenberger, Jeff G.; Bhandari, Aditya Bikram; Dorfman, Kevin D; dorfman@umn.edu; Dorfman, Kevin D.; University of Minnesota Dorfman Research Lab
DNA confinement in a nanochannel typically is understood via mapping to the confinement of an equivalent neutral polymer by hard walls. This model has proven to be effective for confinement in relatively large channels where hairpin formation is frequent. An analysis of existing experimental data for Escherichia coli DNA extension in channels smaller than the persistence length, combined with an additional dataset for lambda -DNA confined in a 34 nm wide channel, reveals a breakdown in this approach as the channel size approaches the Odijk regime of strong confinement. In particular, the predicted extension distribution obtained from the asymptotic solution to the weakly correlated telegraph model for a confined wormlike chain deviates significantly from the experimental distribution obtained for DNA confinement in the 34 nm channel, and the discrepancy cannot be resolved by treating the alignment fluctuations or the effective channel size as fitting parameters. We posit that the DNA-wall electrostatic interactions, which are sensible throughout a significant fraction of the channel cross section in the Odijk regime, are the source of the disagreement between theory and experiment. Dimensional analysis of the wormlike chain propagator in channel confinement reveals the importance of a dimensionless parameter, reflecting the magnitude of the DNA-wall electrostatic interactions relative to thermal energy, which has not been considered explicitly in the prevailing theories for DNA confinement in a nanochannel.
Data from: Measuring the wall depletion length of nanoconfined DNA (2018)
(2018-09-20) Bhandari, Aditya B; Reifenberger, Jeffrey G; Chuang, Hui-Min; Cao, Han; Dorfman, Kevin D; dorfman@umn.edu; Dorfman, Kevin D; Dorfman
Efforts to study the polymer physics of DNA con ned in nanochannels have been stymied by a lack of consensus regarding its wall depletion length. We have measured this quantity in 38 nm wide, square silicon dioxide nanochannels for five different ionic strengths between 15 mM and 75 mM. Experiments used the Bionano Genomics Irys platform for massively parallel data acquisition, attenuating the effect of the sequence-dependent persistence length and nite-length effects by using nick-labeled E. coli genomic DNA with contour length separations of at least 30 m (88,325 base pairs) between nick pairs. In excess of 5 million measurements of the fractional extension were obtained from 39,291 labeled DNA molecules. Analyzing the stretching via Odijk's theory for a strongly con ned wormlike chain yielded a linear relationship between the depletion length and the Debye length. This simple linear fi t to the experimental data exhibits the same qualitative trend as previously defined analytical models for the depletion length but now quantitatively captures the experimental data.
Data from: Sequence-Dependent Persistence Length of Long DNA
(2017-12-05) Chuang, Hui-Min; Reifenberger, Jeffrey G; Cao, Han; Dorfman, Kevin D; dorfman@umn.edu; Dorfman, Kevin D
Using a high-throughput genome-mapping approach, we obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm × 41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5–393 kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk’s theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.
Data supporting 'Subdiffusion of loci and cytoplasmic particles are different in compressed E. coli cells'
(2018-05-15) Yu, Shi; Sheats, Julian; Cicuta, Pietro; Sclavi, Bianca; Cosentino Lagomarsino, Marco; Dorfman, Kevin D; dorfman@umn.edu; Dorfman, Kevin D
The complex physical nature of the bacterial intracellular environment remains largely unknown, and has relevance for key biochemical and biological processes of the cell. While recent work has addressed the role of non-equilibrium drives and crowding, the consequences of mechanical perturbations are relatively less explored.We have used a microfabricated valve system to track both fluorescently labeled chromosomal loci and cytoplasmic particles in E.~coli cells shortly after the application of a compressive force on time scales that are too sudden to allow for biochemical response from the cell. While cytoplasmic diffusion is slowed down significantly under compression, the mobility of DNA loci is much less affected. These results suggest that the dynamics of the bacterial chromosome are decoupled from the viscoelastic environment of the cytoplasm under such short time scales, and that DNA elasticity and nucleoid organization play a more important role in loci subdiffusion than cytoplasmic viscoelasticity.
Did You Give the Government Your Baby’s DNA? Rethinking Consent in Newborn Screening
(Minnesota Journal of Law, Science and Technology, 2014-05) Suter, Sonia
Newborn screening (NBS) has long offered the possibility of identifying rare conditions, which can be lethal or debilitating if not detected and treated quickly in the newborn period. These screening programs, usually mandatory, have been well established in every state since the 1960s. In the last decade, the number of conditions screened for has risen exponentially to include more than fifty inborn errors of metabolism, blood disorders, genetic, or other conditions. Not surprisingly, newborn screening programs have been widely accepted for their potential to save the lives of countless children. Despite their valuable public health benefits, however, old approaches to, and more recent expansions of, NBS raise important privacy and policy concerns. NBS samples are collected in most states without affirmative, or sometimes any, consent from parents. NBS programs now screen for an ever-broadening range of diseases—sometimes without careful assessment of the risks and benefits—including conditions for which there is no treatment. NBS samples are retained for long periods or indefinitely. And finally, few, if any, limits prevent potentially invasive uses of these samples by the government or third parties. Indeed, evidence suggests that a great deal of research is being conducted on these stored blood spots, the collection and storage of which many parents are simply unaware. Only a few lawsuits and legislatures have addressed the legality of these practices. With recent expansions in the scope of NBS and increased interest in these samples for research, it is time to take a fresh look at this long-standing public-health system and to reexamine some of the underlying philosophies and practices associated with it. While NBS offers important public health benefits, it also threatens some of the civil liberties of the parents and children involved. This piece argues for the need to strike a careful balance between the public goods and private interests, and describes a methodology that allows these competing values to be recognized in policymaking. It concludes by suggesting ways to balance the important values of maximizing the well-being of newborns and promoting research, while also protecting autonomy and privacy as much as possible.
DNA confined in nanochannels and nanoslits
(2014-05) Tree, Douglas
It has become increasingly apparent in recent years that next-generation sequencing (NGS) has a blind spot for large scale genomic variation, which is crucial for understanding the genotype-phenotype relationship. Genomic mapping methods attempt to overcome the weakesses of NGS by providing a coarse-grained map of the distances between restriction sites to aid in sequence assembly. From such methods, one hopes to realize fast and inexpensive de novo sequencing of human and plant genomes.One of the most promising methods for genomic mapping involves placing DNA inside a device only a few dozen nanometers wide called a nanochannel. A nanochannel stretches the DNA so that the distance between fluorescently labeled restriction sites can be measured en route to obtaining an accurate genome map. Unfortunately for those who wish to design devices, the physics of how DNA stretches when confined in a nanochannel is still an active area of research. Indeed, despite decades old theories from polymer physics regarding weakly and strongly stretched polymers, seminal experiments in the mid-2000s have gone unexplained until very recently.With a goal of creating a realistic engineering model of DNA in nanochannels, this dissertation addresses a number of important outstanding research topics in this area. We first discuss the physics of dilute solutions of DNA in free solution, which show distinctive behavior due to the stiff nature of the polymer. We then turn our attention to the equilibrium regimes of confined DNA and explore the effects of stiff chains and weak excluded volume on the confinement free energy and polymer extension. We also examine dynamic properties such as the diffusion coefficient and the characteristic relaxation time. Finally, we discuss a sister problem related to DNA confined in nanoslits, which shares much of the same physics as DNA confined in channels.Having done this, we find ourselves with a well-parameterized wormlike chain model that is remarkably accurate in describing the behavior of DNA in confinement. As such, it appears that researchers may proceed with the rational design of nanochannel mapping devices using this model.
DNA-protein cross-linking by bifunctional DNA alkylating agents.
(2010-03) Michaelson-Richie, Erin Denise
Many common DNA alkylating agents, such as environmental toxins and chemotherapeutic drugs, are bis-electrophiles capable of covalently cross-linking cellular biomolecules. While DNA-DNA cross-linking by such compounds is well-characterized, the identities and the biological effects of the corresponding DNA-protein cross-links (DPCs) are poorly understood. Furthermore, because bis-electrophiles produce DNA-DNA cross-links and DNA monoadducts in addition to DPCs, it is difficult to establish the biological outcomes specifically resulting from DPC lesions. The purpose of the present work was to characterize DNA-protein cross-linking by two bis-electrophiles, 1,2,3,4-diepoxybutane (DEB) and bis(2-chloroethyl)methylamine (mechlorethamine), and to evaluate the ability of DPCs to induce cytotoxic and mutagenic effects. Mass spectrometry-based proteomics and immunological detection methods identified 41 proteins participating in DPC formation in the presence of DEB in nuclear protein extracts prepared from human cervical carcinoma (HeLa) cells, and 38 proteins which formed DPCs to the chromosomal DNA of human fibrosarcoma (HT1080) cells treated with mechlorethamine. Relative to their cellular abundance, a disproportionately high number of the proteins involved in DPC formation were nuclear proteins with known nucleic acid-binding capabilities which participate in cellular processes such as transcriptional regulation and DNA repair. HPLC-ESI+-MS/MS analysis of total proteolytic digests of DPCs revealed the chemical structures of the cross-links produced by DEB and mechlorethamine to be 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) and N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA), respectively. In order to analyze the biological consequences of DPC lesions, we selectively induced DPCs in mammalian cell cultures by electroporating them in the presence of epoxide-containing protein reagents. Significant levels of cell death and mutations were observed, suggesting that DPC lesions contribute to the biological effects of bis-electrophiles.
Dynamics of Knotted DNA Molecules Confined in Nanochannels
(2022-03) Ma, Zixue
Knots are intriguing topological objects and ubiquitous in biopolymers such as DNA molecules. The occurrence of knots in DNA confounds the accuracy of genomics technologies, such as nanochannel-based genome mapping and nanopore sequencing, that require uniformly stretching the DNA molecules. Knots existing in vivo also influence biological processes, such as DNA replication, and hence leads to cellular malfunction. The control of DNA knots is, thus, significant for genomics technologies and cell survival, which require first understanding the fundamental properties of knotted DNA in a crowded environment. The aim of this thesis is to address fundamental questions related to knot transport in nanochannel-confined DNA molecules, particularly the knot diffusion mechanism, the effect of knots on DNA diffusion in nanochannels and the interactions between two knots. We first determined the knot diffusive behavior along DNA confined in nanochannels to distinguish between two predicted knot diffusion mechanisms, self-reptation and knot region breathing. With a recently developed nanofluidic "knot factory" device, we generated knots in DNA molecules efficiently. The experimental results of knot motion along DNA chains show that knots undergo subdiffusion, i.e. their mean-squared displacement grows sublinearly with time, which supports the knot diffusion mechanism of self-reptation. We then investigated the effects of knots on DNA center-of-mass diffusion in nanochannels, thus resolving the open question which of these competing effects, the shortening of DNA chains or the increased DNA-wall friction, dominates knotted DNA diffusion in nanochannels. To address this question, we measured the diffusivity of DNA molecules before and after knot formation via a combination of the nanofluidic knot factory device for knot generation and laser-induced fluorescence microscopy for DNA observation. The experimental results show that the presence of knots decreases the diffusivity of DNA chains confined in nanochannels. The reduced diffusivity indicates that the DNA-wall friction, rather than the shortening of the confined chain size, dominates the friction of knotted DNA in nanochannels. Our previous work focused on the dynamical properties of single knots. Long DNA molecules are susceptible to form multiple knots in the chains. In the third research project, we investigated the interactions between two knots in nanochannel-confined DNA by analyzing the motion of the two knots along DNA chains. The free energy profiles of knot-knot interactions show that the separated knot state is more stable than the intertwined knot state, with dynamics in the separated knot state that are consistent with independent diffusion of the two knots. The thesis work provides deep insights into the dynamical properties of DNA knots under nanochannel confinement. We hope such fundamental knowledge gained in this dissertation could prescribe avenues for suppression and removal of knots under nanofluidic systems and crowded environments, thereby improving the genomic technologies and controlling knots in living cells.
Eeffective pDNA and CRISPR RNP delivery promoted by design of cationic bottlebrush and combinatorial polymers synthesis
(2022-07) Dalal, Rishad
The field of gene therapy has grown in response of the millions of people who suffer from genetic diseases worldwide. As genetic payloads need a delivery carrier, cationic polymeric vectors have grown in promise as delivery vehicles that are more cost-effective, scalable, and stable in comparison to viral vectors. The field of polymeric gene delivery has focused on improving delivery efficiency through chemical and structural modifications. Herein, we have made steps towards understanding how architectural modifications and how structure property relationships can improve the field of gene delivery. Initially it was found that when comparing cationic homopolymers, a bottlebrush architecture outperformed a linear analog in over pDNA delivery efficacy. Follow-up studies explored how bottlebrush end-group hydrophilicity can play a role in balancing colloidal stability, gene expression, and cellular viability. In addition to architectural understanding, studies to understand how structure-property relationships within linear polymers were explored in which a combinatorial library of 36 polymers was synthesized and used to deliver pDNA and CRISPR-Cas9 RNP. Machine learning aided in optimizing and analyzing structural relationships relative to expression outputs. Overall, we were able to create guides in improving gene expression through the optimization of polymer macromolecular structure and unique chemical understanding per biological payload.
Electrophoresis of large DNA with a sparse zinc oxide nanowire array.
(2010-05) Araki, Noritoshi
We developed a simple inexpensive method to integrate ZnO nanowires into a microchannel using a combination of aqueous solution synthesis of ZnO nanowires and photolithography, which is used as a nanowire-embedded microfluidic device. The density of ZnO nanowires inside the microchannel is controllable by simply changing the concentration of the seed solution. We conducted a study of dynamic interactions between electric field driven !DNA and a single isolated ZnO nanowire using single molecule spectroscopy technique. The study shows that the hooking time is exponentially dependent on b/Rg, in agreement with a prediction by simulation work. We also find that the hooking probability for small values of b/Rg increases as the electric field strength increases.
Enhanced Efficacy of Plasmid DNA Vaccines for Cancer Therapy
(2010-11) Dietz, Wynette
Since cancer is the second most common cause of death in the United States, it is of great importance to pursue new and improved methods for treating cancer. The goal of cancer immunotherapy is to exploit the specificity and longevity of immune responses through the use of vaccines to treat cancer. DNA vaccines have many advantages over protein and viral vaccine-based strategies including low cost, ease of production, flexibility and low toxicity. Plasmid DNA vaccines encoding tumor antigens can produce powerful anti-tumor immune responses in animal models, but clinical trials have shown only modest responses. This lack of clinical efficacy is thought to reflect the two major limitations of plasmid DNA vaccines: transient protein expression and low transfection efficiency. Transient protein expression is likely the result of gene silencing due to transcriptionally repressive chromatin within the plasmid backbone. To overcome this limitation, we removed the bacterial backbone sequences and produced a minicircle DNA consisting of the gene expression cassette with only a few bases of the bacterial backbone. This resulted in persistent protein expression, increased transfection efficiency and enhanced immunogenicity. In an effort to further enhance the transfection efficiency, we produced cationic carriers that bind plasmid DNA and protect it from degradation. The addition of these cationic carriers significantly increased transfection efficiency in vitro but has yet to show the same effect in vivo. Additionally, we administered these vaccines transdermally using a tattoo device and achieved rapid and potent immune responses. Our results suggest transdermal delivery of a minicircle DNA vaccine elicits potent antigen-specific immune responses, and as such, holds great promise for cancer therapy. Future work will include increasing transfection efficiency in vivo and increasing the immunogenicity of the vaccines through the use of adjuvants with the goal of producing feasible, efficacious DNA vaccines for cancer therapy.

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