University of Groningen Applications of DNA hybrids in biobased medicine and materials Liu, Qing

Applications of DNA hybrids in biobased medicine and materials

Liu, Qing

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Chapter 1

Hydrophobic Modification of DNA

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1.1 Introduction

As one of the most studied biomacromolecules, deoxyribonucleic acid (DNA) has fascinated researchers not only by its role as a carrier for the genetic information but also its utilization as a structural motif for the assembly of nanostructures, which is termed DNA nanotechnology. Thanks to the introduction of automated solid phase synthesis, molecular cloning techniques and polymerase chain reaction, a greater range of studies involving oligonucleotides and long nucleic acid strands became possible at an affordable price and effort.

The profound interests upon DNA could be attributed to its unique properties, including self-recognition, sequence programmability, robustness and moderate resistance to degradation. In 1991, an artistic building strategy for artificial DNA structures using pristine DNA was introduced by Seeman.1 _{A series of DNA}

architectures have been realized ever since, ranging from two-dimensional arrays 2-5 _{to well-defined three dimensional tubes,}6 _{convex polyhedra}7-11 _{and brick and}

origami structures.12-15 _{The decisive factor behind all these designs is the proper}

persistence length of a double-stranded (ds) DNA (50 nm). Due to their well-defined size and shape, significant progress has been made in various application areas employing such DNA nanostructures that range from nanoscale assembly line,16

drug delivery,17,18 _{nucleic acid detection,}19 _{and DNA machines.}20,21

Additionally, great efforts have been devoted to developing applications of DNA by combining its information-carrying capability with new functionalities introduced by new units. Thanks to the enormous development in DNA technology, a great range of tailor-made modifications are commercially available nowadays for daily laboratory usage, which in return promotes the advance of DNA technology. For example, when an acrydite is attached to DNA, DNA-based gels could be obtained upon polymerization, which show appealing performance in sensors,22 _{drug release}

scaffolds23 _{or as matrices for the triggered activation of enzyme cascades.}24 _In

addition to the introduction of new functionalities to DNA, ongoing research has also been focusing on altering molecular structures, morphology, self-assembly properties or even solubility of DNA hybrids upon the introduction of tailored structural units. These can be materials being composed of small organic molecules,25 _polymers,26-28 _{inorganic nanoparticles that are covalently attached to}

DNA29-31 _{or surfactants that are combined through electrostatic interactions.}32-33

The first class of DNA hybrids tends to assemble into nanoparticles with a hydrophilic DNA corona and an organic or inorganic core. Such hybrids have found use in DNA detection,34 _{gene regulation,}35 _{template synthesis}36 _and

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charged surfactants, is prone to aggregate and precipitate from aqueous phase. These DNA/surfactant complexes have also been extensively studied and found vast applications.38-41

In recent years, a range of hydrophobic structures have been applied to modify DNA. These DNA hybrid materials either tend to self-assemble into micellar systems with dimensions on the nanoscale in aqueous phase due to micro phase separation or precipitate from the aqueous phase. Their behaviors are very different from pristine DNA. Hence, these materials present appealing potential for applications in the fields of bionanotechnology and nanomedicine. In this chapter, the hydrophobic modification of DNA through small organic molecules, polymers and surfactants will be briefly reviewed.

1.2 Modification Methods

1.2.1 Solid-Phase synthesis (SPS) of nucleic acids

Figure. 1.1. Hydrophobic modification of (A) 5’- and (B) 3’- end of DNAs using solid-phase synthesis. (a) deblocking of DMA; (b) coupling of activated CEPA to 5’-end; (c) standard synthesis with nucleoside phosphoramidite.

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In 1955 the first dinucleotide was chemically synthesized.42 _{However, a universal}

method for the chemical preparation of both DNA and RNA was not reported until 1980 in which 2’-deoxynucleoside-3’-phosphoramidites were used as synthons.43,44

The SPS technique now makes it possible to synthesize short fragments of DNA (up to 200 mers). At the same time, researchers also found it convenient to introduce functional groups to DNAs while the DNAs are still attached to the solid support. One classic method consists of using 2-cyanoethyl-N, N-diisopropylphosphoramidite (CEPA) groups to introduce hydrophobic structures to DNA.45 _{The hydrophobic}

molecule is first activated with CEPA and then attached to the 5’-end of DNA on the solid supports (Figure 1.1 A). The coupling to the 3’-end of DNA could be achieved either through reverse synthesis employing special CEPA activated units and final attachment of the hydrophobic moiety (similar to coupling to 5’-end in Figure 1.1 A) or using custom solid supports, which already contain the desired hydrophobic moieties that is connected via a cleavable linker (e.g. carboxylic ester) (Figure 1.1 B).46

With SPS, the coupling of hydrophobic entities to DNA could be done either with or without a DNA synthesizer. With a DNA synthesizer, the full synthesis including the hydrophobe can be regarded as a fully automated process. The advantages of the fully automated synthesis are precise control and monitoring of the entire procedure, higher reproducibility and relatively large scales. A wide range of hydrophobic units have been successfully attached to DNAs within a DNA synthesizer.47-50 _{Very recently, the automated synthesis technique was employed to}

prepare polymers and DNA-polymer conjugates through atom transfer radical polymerization (ATRP).51

The coupling of the hydrophobe without a DNA synthesizer is often used to circumvent the shortcomings of fully automated synthesis. In this way, the hydropbobe is often coupled using a syringe or in a flask with DNA on solid supports as synthesized.This method can be specifically tailored to satisfy the requirements of some synthesis, which cannot be achieved on a synthesizer, like incompatible solvents or catalysts, extreme reaction conditions or longer reaction time.

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1.2.2 Solution coupling

Figure. 1.2. Illustration of the organic phase synthesis of hydrophobic molecules modified DNAs.

Solution-phase coupling of DNA with hydrophilic molecules in an aqueous environment has been demonstrated to be highly versatile and efficient at various positions of the DNA molecule. It is a very widely used method to functionalize DNA with groups like amines or thiols and to attach those to specific complementary functionalities like carboxylic acid, thiols or maleimides. Modification of DNA with hydrophobic molecules in aqueous phase, however, is less efficient due to solvent incompatibility of the two components i.e. the nucleic acid is soluble in water while the hydrophobe is not.52

Recently it was reported in our group that with the help of positively charged surfactants, hydrophobic molecules could be attached to DNA even in organic phase, as shown in Figure 1.2.53 _{In short, terminal functionalized DNAs (e.g. amine) were}

first complexed with positively charged surfactants, precipitated and freeze-dried. The resultant DNA/surfactant complexes are not soluble in aqueous phase but can be dissolved in organic solvents like DMF, CHCl3 and THF while the surfactants do

not compromise the reactivity of the functional group on the DNA. Therefore, the coupling of hydrophobic molecules to DNA could be carried out in organic phase with high efficiency. After the reaction, the surfactants were removed by simply washing the solutions with brine, yielding the desired products. Pyrene, hydrophobic alkyl chains and polystyrene were attached to the terminals of DNA in high yields.

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1.2.3 Complexation with surfactants

Figure. 1.3. Schematic representation of four representative LLC structures of dsDNA-lipid complexes: (a) lamellar phase; (b) and (c) hexagonal phase; (d) cubic phase.

Hydrophobic modification of DNA could be realized by complexation with positively charged surfactants through electrostatic interactions, yielding water-insoluble DNA/surfactant complexes. Their properties are different from the ones from DNA amphiphiles. These complexes consist of a DNA backbone from which surfactants protrude as non-covalently bonded side chains. Their formation is electrostatically driven and they form bulk films, lyotropic as well as thermotropic liquid crystals and hydrogels. Owing to their molecular shape and weak intermolecular interactions, such as van der Waals and dipolar forces,54-56 _{these complexes tend to}

spontaneously form highly organized assemblies like ordered lamellar, hexagonal and cubic structures as well as disordered isotropic phases,57,58 _{as shown in Figure}

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1.3 Applications

DNA modified with hydrophobic molecules tends to self-assemble into micellar systems with nanometer dimensions because of micro phase separation (except for DNA/surfactant complexes). These nano-sized particles are composed of a soft hydrophobic core and a hydrophilic shell, which has made them very promising candidates in fields like drug delivery, DNA detection and template reactions. On the other hand, DNA/surfactant complexes are mainly investigated in bulk film or as liquid crystals. In both states the respective materials exhibit well-defined structures, high structural stability and easy processability, thereby allowing diverse applications ranging from optoelectronics to biomedicine and therapeutics.

1.3.1 Drug delivery

Figure. 1.4. (a) Schematic representation of PPO-b-DNA copolymer micelle: folic acid (red dots) modified complementary oligonucleotides are hybridized to functionalize the surface of the nanoparticle with targeting units and drugs (green dots) are introduced to the core of the nanoparticle through mixing. (b) Schematic representation of PPO-b-DNA copolymer and Pluronics hybrid micelle. (A) PEG block of Pluronic; (B) DNA block of DBC; (C) PPO blocks of Pluronic and DBC; (D, E) Probes at 5’- and 3’- ends of the cDNA, respectively. (F) Hydrophobic compound loaded into the hydrophobic core. (G) Cross-linked nanodomains of PETA in the core.

Due to the self-recognition properties of the DNA part and the presence of a hydrophobic core to serve as a carrier unit, DNA amphiphiles have been extensively

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studied for drug delivery. With polypropylene oxide-DNA block copolymer (PPO-b-DNA) micelles, researchers in our group exploited it in cancer therapy.59 _{It was}

found that the shell functionality of the nanoparticles could be easily introduced through hybridization with functionalized complementary oligonucleotide (in this case, folic acid) while the hydrophobic interior could be loaded with hydrophobic anticancer drugs through simple mixing (Figure 1.4 a). Cell culture experiments with Caco 2 cells showed that these nanoparticles could be taken up through receptor mediated endocytosis and resulted in efficient cytotoxicity and high mortality. Later a more sophisticated design was proposed in our group (Figure 1.4 b).60 _{In this design, Pluronics, a triblock copolymer with a PEG–b-PPO–b-PEG}

architecture, was blended with PPO-b-DNA copolymer. As a result, the PPO from both DNA copolymer and Pluronics formed the core of micelles while DNA from copolymers and PEG from Pluronics were located in the corona. The PEG part did not undermine the hybridization of DNAs while the hydrophobic core could be loaded with hydrophobic drugs or cross-linked to prevent dissociation upon dilution.

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Changing the environment from water to bulk materials, DNA-surfactant complexes are characterized by long term in vivo stability of the DNA component and subsequently can recover biological activity once wetted.61 _{These structures contain}

a high local concentration of the therapeutic nucleic acid agent and hence the study of bulk DNA-surfactant structures is developing into an exciting field for drug delivery applications. Tirrell and co-workers recently simulated a drug release system by employing an ethidium bromide-labelled DNA-didodecyldimethylammonium bromide (DNA-DDAB) film.62 _{When exposed to a}

buffer solution, such as PBS, electrostatic screening of the DNA-complex charges led to disassembly of the film in a layer-by-layer fashion releasing ethidium bromide (Figure 1.5). It is thus reasonable to expect the film to disassemble easily in vivo and to act as a drug delivery vehicle or DNA depot. Similarly to ethidium bromide, anticancer drug molecules, such as daunorubicin and doxorubicin, also intercalate into DNA inhibiting the enzyme topoisomerase II and preventing the DNAs from

18

duplication in fast-replicating cells.63 _{These drugs are potentially useful in}

DNA-surfactant films as they could be incorporated into the complex and then locally elute after implantation upon film degradation, either through nuclease action or ion replacement. In this fashion, a drug delivery system was realized by blending the DNA-surfactant complex with poly (lactide-co-glycolide) and the drug daunorubicin.64 _{The complex was obtained by casting a film from DMSO/CHCl3}

solution and subsequent incubation into an aqueous solution of daunorubicin with drug loading being controlled by the immersion time. The pharmaceutically active payload was released from the films after introduction into PBS and the release rate was dependent on the chemical structure of the surfactants. These results clearly indicate that a film of the three components – DNA, surfactant and PLGA – is a promising matrix for anticancer drug delivery.

1.3.2 Liposome decoration

Liposomes are a class of nanocontainers which are able to encapsulate and protect both small molecules and bio-macromolecules, such as protein and DNA. They are made of two layers of amphiphilic lipid molecules of which the hydrophilic heads face to the aqueous solution while the hydrophobic tails point inwards towards each other.65 _{Liposomes play an important role in the physiology and pathophysiology of}

living systems and they have been intensively studied to understand and manipulate biological events and processes.66-68 _{Considering the similarity between liposomes}

and DNA amphiphiles, it is reasonable to predict that DNA amphiphiles can play a role in decorating liposomes.

A. R. Pulido designed a selective cargo-release system with the help of PPO-b-DNA,69

as shown in Figure 1.6. In this design, PPO-b-DNA was first inserted into the surface of vesicles with DNA pointing to the aqueous phase, tagging these nanocontainers with sequence information. Next, a BODIPY monoiodine (BMI) photosensitizer modified complementary DNA was used to hybridize with the DNA on the vesicle surface, which subsequently induced the generation of singlet oxygen close to the lipid membrane under light irradiation. The resulting oxidation of the PPO chains or highly unsaturated phospholipids effectively mediates liberation of the vesicle payload.

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Recently, it was reported by our group that hydrophobicity could be imparted to DNA through the introduction of a dodec-1-yne chain at the 5-position of a uracil base, which was subsequently coupled to DNA by solid phase synthesis, termed lipid-DNA.70_{With this method, the position and number of hydrophobic uracils}

could be chosen with a similar efficiency as normal bases. The lipid-DNA was successfully utilized to induce vesicle fusion.71 _{This current work deals with a}

strategy for anchoring oligonucleotides on a membrane by lipid-modified nucleobases rather than by attaching hydrophobic units to the 3’- or 5’-termini. Single-stranded DNAs functionalized with four lipid-modified nucleobases were stably grafted onto the membrane of lipid vesicles. The orientation of DNA hybridization and the number of anchoring units played a crucial role in liposomal fusion, which in the most efficient system reached remarkable 29% content mixing without notable leakage.

Besides, DNA amphiphiles have been employed to realize the assembly of larger containers,72 _{mimicking cellular systems}73 _{and gene silencing.}74

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1.3.3 Nanoscience

DNA amphiphiles are not only suitable for the construction of nanoscopic structures like spherical or cylindrical micelle aggregates or vesicles but they can be manipulated using DNA.

With PPO-b-DNA, scientists were able to control the size and geometry of the structures.75 _{As shown in Figure 1.7 a, 22mer DNA coupled with PPO (Mw=6800}

g/mol) self-assembled into spherical particles with a hydrodynamic radius of 10nm. When the number of bases was enzymatically extended to 84, particles with radius of 23nm were obtained. The geometry of the particles can be modulated through hybridization with a long complementary DNA template, which resulted in rod-like micelles.

Figure. 1.7. (a) Schematic representation and AFM images of the morphology change of PPO-b-DNA amphiphiles upon hybridization with short PPO-b-DNAs (top) and long PPO-b-DNAs (bottom). (b) Schematic representation and TEM images of (i) spherical particles from initial DNA-brush copolymers, (ii) rod-like structure formation after the addition of phosphodiesterase enzyme and (iii) morphology change of aggregates upon the addition of different DNA sequences.

Another elegant design involved phosphodiesterase enzyme.76 _DNA-brush

copolymer amphiphiles were obtained by grafting DNAs to a hydrophobic copolymer. Long DNA side chains gave the copolymers higher surface curvature, which resulted in spherical aggregates. When the DNA was cut by an enzyme, a rod-like morphology was formed. Later, upon hybridization with partial complementary DNA to the side chains, spherical particles were formed again because of the increased surface curvature caused by elongated DNA chains. The partial complementary DNA could be removed by adding a DNA which is fully complementary to it, resulting in amphiphiles with short DNAs hence rod-like aggregates. Upon changing DNA sequences, a reversible control over the morphology of the aggregates was realized. (Figure 1.7 b)

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In a different design, hydrophilic moieties were attached to the outside of DNA nanostructure. Thereby, a DNA cube scaffold was preassembled with single-stranded regions, which were further hybridized with four polymer modified DNA strands, as shown in Figure 1.8.77 _{The polymer was located at various corners of the}

cube, which increased the nuclease resistance comparing to DNA cubes without polymers.

1.3.4 DNA detection

Similar DNA side chain polymers that were described in the previous paragraph were employed also for DNA detection. When several DNA strands are grafted along one polymer chain the melting curves are sharper and higher when hybridized with complementary DNA compared to pristine DNA.78 _{This characteristic melting}

behavior was exploited for DNA detection. In one study, DNA as well as ferrocene units were grafted to a polymer backbone and the resulting hybrids were used in a sandwich-type electrochemical detection strategy (Figure 1.9).79 _{A probe sequence}

was first immobilized on the surface of a gold electrode. After that, the hybrids and target DNA were added and the redox signal was measured. The sensitivity of this strategy reached 100 pM. DNA-grafted polymer based DNA detection has also be exploited in several other studies.80-82

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Figure. 1.9. Schematic representation of DNA detection employing a DNA and ferrocene grafted polymer.

1.4 Thesis Motivation and Overview

Tremendous efforts have been devoted to the development of DNA functionalization methods over the last decades, including chemical modification and physical complexation. With specific modification, tailored functionalization and potential applications could be realized. For example, when coupled with a fluorophore, DNA could be used to detect particular sequences. When a hydrophobic structure was coupled to DNA, it is possible to yield either DNA amphiphiles or hydrophobic DNA hybrids. Nevertheless, both DNA architectures present some unique properties that pristine DNA does not have. These features were important to successfully realize potential applications in the context of dynamic DNA assemblies, drug delivery and DNA detection. With a great advancement already existing in DNA functionalization techniques, we are seeking easier and more applicable ways as supplements to current ones and exploiting new applications out of it. This thesis focuses on new properties and potential applications of hydrophobically functionalized DNA. The first Chapter briefly introduced the development of hydrophobic modification of DNA, including chemical coupling and physical complexation. Their self-assembly properties and applications were highlighted. Despite the many examples that have been listed, new exciting discoveries in DNA functionalization are awaiting to be exploited.

Dodec-1-yne chain-modified DNA (lipid-DNA) was one of the DNA amphiphiles that were developed in our group and found vast applications. In Chapter 2 a new class of lipidated aptmer based functional structure were used for the catalytic oxidation of dopamine to aminochrome, which expanded the application of lipid-DNA. Lipidated hemin/G-quadruplex (hGQ) units and the lipidated dopamine binding aptamer (DBA) units were first synthesized and assembled into micellar

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nanostructures. Then the critical micelle concentrations (CMC) for the different ratio of lipid-DNA were measured. Subsequently, the catalytic functions of the micelles toward the H2O2-mediated oxidation of dopamine to

aminochrome were evaluated. Besides, a fully synthetic lipidated catechol oxidase-mimicking dinuclear Cu(II)–BPMP complex was also synthesized and employed to explored the possibility of substituting the lipidated hemin/GQ catalytic sites in the micelles.

In the following chapter, lipid-DNA was tested in the context of ophthalmic drug delivery. Nowadays, low efficiency of topical drug delivery formulations is an acknowledged problem in ophthalmology. To address this challenge, in Chapter 3, we introduced lipidated aptamers (lApts) nanoparticles (NP) as a new class of vehicles for ophthalmic drug delivery. The generality of this approach was demonstrated employing three aptamers selected for the ophthalmic therapeutics: travoprost, brimonidin and kanamycin B. In aqueous solution the synthesized lApts formed nanosized micelles with a hydrophobic core and a hydrophilic aptamer corona. The loading capacity of aptameric corona of the NP and the effect of the lipid modification on aptamer ligand binding characteristic were tested. Subsequently, the cornea binding properties and toxicity of the lApts were evaluated ex vivo and in

vivo. Finally, the efficacy of lApt NPs in antibiotic delivery to animal tissue is

investigated.

Externally regulated biomacromolecules are now considered as particularly attractive tools in nanoscience and the design of smart materials, due to their highly programmable nature and complex functionality. Incorporation of photoswitches into biomolecules, such as peptides, antibiotics and nucleic acids, has generated exciting results in the past few years. Molecular motors offer the potential for new and more precise methods of photoregulation due to their multistate switching cycle, unidirectionality of rotation, and helicity inversion during the rotational steps. In

Chapter 4, we designed and synthesized a photoswitchable DNA hairpin, in which

a molecular motor serves as the bridgehead unit. With the photoswitchable bridgehead in place, hairpin formation was checked and photochemical properties of the motor part in this advanced biohybrid system were evaluated. Rotation of the motor generates large changes in structure, and as a consequence the duplex stability of the oligonucleotide could be regulated by UV light irradiation. The results presented herein establish molecular motors as powerful multistate switches for application in biological environments.

In Chapter 5 hydrophobic modification of DNA was extended to physical complexation with positively charged surfactants. A new class of DNA-surfactant

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based organogels by simply mixing DNA with cationic surfactants and subsequent immersion in organic phases. It was found that these organogels showed excellent absorbability of organic solvents. POM and SAXS tests demonstrated a liquid crystalline state of these organogels. Additionally, mechanical properties, processability and self-healing property of these organogels were evaluated. Upon with the introduction of iron oxide nanoparticles, the liquid crystal phase of DNA-surfactant organogels were not compromised but a magnetic response to the organogels was endowed. Hence, with these properties, DNA-surfactant LC organogels may expand the application of organogels to new potential applications.

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