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Development, Practice, and Perspectives for DNA Enzyme Systems

Review

Development, Practice, and Perspectives for DNA Enzyme Systems

Kevin MacVittie1 , Petra Kraus2*

1Department of Chemistry & Biomolecular Sciences, Clarkson University, Potsdam, NY 13699

2Department of Biology, Clarkson University, Potsdam, NY 13699

*Corresponding Author, E-mail: pkraus@clarkson.edu

Abstract

DNAzymes are still fairly recent man-made additions to the growing spectrum of synthetic molecules dramatically advancing the field of biomolecular science. These catalytically active nucleic acids have great potential in personalized medicine and as internal biosensors owing to their low immunogenicity, as well as in bio-computing, because of their robust nature and large data storage capacity. Their future potential is evident in areas that range from the development of biofuel cells to power implanted electronics, like pacemakers, where they could exceed capabilities and circumvent obstacles of existing protein-based catalysis.

Keywords

DNAzyme, biosensor, bio-computing, catalytic nucleic acid, biofuel cell

Introduction

Since their synthesis in 1994 [1] DNA-based enzymes have evolved into an exciting area of biochemistry, molecular biotechnology and bio-computing. The catalytic activity of short stranded nucleic acids was first discovered over thirty years ago when naturally occurring ribonucleic acid (RNA) molecules were discovered to act as catalysts in reactions such as phosphodiester bond hydrolysis, thus named ribozymes [2]. Translation of RNA into protein is accomplished by a ribozyme in the active center of cellular ribosomes [3]. Most RNAs are relatively unstable molecules, as their intended use only requires short lifespans in vivo, while their transcriptional origin, the deoxyribonucleic acid (DNA) is far more robust, rendering DNA a more suitable molecule for most approaches [1].

As known to date, nature has not generated catalytically active DNA in vivo to the extent it has with RNA-based ribozymes [1], yet Mankind succeeded in developing catalytically active short stranded oligonucleotides termed deoxyribozymes or DNAzymes synthetically [1]. While DNAzymes are currently not utilized in commercial applications, the fundamental research behind their capabilities is abundant [1,2,4-6]. The primary use for DNAzymes lies in the manipulation of nucleic acids, whether it be their cleavage [1] or ligation [4]; however, more atypical uses have been found, including the reaction of metal ions with porphyrin rings [7]. Much like traditional protein enzymes, DNAzymes exhibit a high level of selectivity and specificity. This is intrinsically a result of their construction from nucleic acids: small molecules notorious for their exceptionally elevated specificity and selectivity to their given conjugate base pair [8].

DNAzymes present advantages over traditional enzymes. DNA is well-known for its long-term stability at a wide range of temperatures and is even considered an attractive alternative for long-term data storage [9], whereas protein-based enzymes are typically more sensitive to fluctuations in temperature, and could undergo undesired conformational changes or denaturation. Furthermore the ability of DNA to be stored in a dry state allows for a much higher level of convenience when dealing with the transportation and distribution of DNAzyme-based technologies [10]. Following this logic, DNAzymes have been immobilized on electronic transducers [11] and nanoparticles [12] for use in detection systems, for bio-computing architectures and for their use as molecular switches in signal amplification [13].

The lack of naturally occurring DNAzymes in vivo, requires their synthesis in vitro. However, prior to mass-production of any DNAzyme, a desirable catalytic activity and target specificity must be confirmed. Therefore, libraries with large populations of fixed-length random sequence oligonucleotides are generated and screened for their interaction with a compound of interest via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [8]. Purified ligated compounds are typically isolated and amplified by PCR to generate a large quantity of highly specific DNAzymes [14]. Recently it has been shown that enzyme function can be achieved by molecular in vitro evolution from a non-catalytic sequence, leading to a DNAzyme with RNA cleavage properties [15].

Applications of DNAzymes

DNAzymes in Cancer Treatment

Since their development, DNAzymes have been used for a variety of applications. One of their greatest potentials within the medical research community is the manipulation and silencing of RNA pathways for the inhibition of gene expression, typically for use in cancer therapy. With this in mind, a “general purpose” RNA-cleaving Mg2+ dependent DNAzyme was synthesized [16]. This DNAzyme, called 10-23 DNA Enzyme, consists of two substrate-recognition domains of approximately eight bases separated by a 15 nucleotide catalytic domain. The domain-recognition “arms” can be tailored to any set of complimentary RNA bases, resulting in the catalyzed cleavage of mRNA. In this context, a promising use for DNAzymes could lie in targeting osteosarcoma at the source [17]. Osteosarcoma is a debilitating cancer typically weakening the bones of adolescents and young-adults and commonly metastasizing to the lungs [18]. Most cases of high-grade osteosarcoma are found to have increased expression of c-jun, a proto-oncogene [19,20]. The DZ13 DNAzyme catalyzed gene silencing could stimulate apoptosis of osteosarcoma cells by the down-regulation of c-jun mRNA [18,19,21,22], while having no adverse effect on cell proliferation of non-osteosarcoma cells. The first clinical trial in humans for the treatment of basal cell carcinoma (BCC) with DZ13 successfully completed Phase 1, showing decreased expression of c-jun in patients with nodular BCC, while no drug-related serious side effects were reported [23]. These promising results could pave the way for the use of DNAzyme DZ13 as a cancer treatment drug for controlled apoptosis of cancer cells with obvious and significant implications for future cancer treatments [17].

DNAzymes in Bio-computing Systems

Aside from their application in cancer therapy, DNAzymes have been used extensively in the realm of bio-computing. Contrary to popular belief, a vast majority of bio-computing does not aim to compete as an alternative to modern silicon computing. Rather, bio-computation focuses on the development of architectures for interfacing existing computing motifs with living systems in biological environments. This is typically in the form of chemical and biochemical interface systems that can directly interact with biomolecules, but also communicate with digital systems through optical or electric measurements [24]. One line of focus lies in the creation of systems capable of performing various Boolean logic operations, from relatively simple realizations such as AND and OR [25-28] to more complicated architectures including XOR, NOR, NAND, INHIB, and XNOR [29-42]. These Boolean logic motifs can then be integrated into larger autonomous “physio-diagnostic” devices, capable of real-time monitoring of living systems with built-in logic [43].

These bio-computing devices are often realized using enzymes, and similar biomolecules capable of direct interaction with biological systems [44-47]. Horseradish peroxidase (E.C. 1.11.1.7) is an enzyme frequently used in bio-computing owing to its ability to oxidize common redox species and to participate in colorimetric reactions [48]. Therefore, it is logical that this would be one of the first characteristics that researchers attempted to mimic using DNAzymes. This was successfully done with the development of a guanine-rich DNAzyme, capable of complexing with a hemin molecule for the catalysis of the oxidation of common redox species, such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), in the presence of hydrogen peroxide [49,50]. When presented with a specifically programmed mRNA scaffold and a hemin molecule the activity of this DNAzyme was activated, allowing for the controllable oxidation of photoactive redox dyes, such as ABTS. This is essential to bio-computing, because almost all bio-computation motifs include a final step resulting in the controlled oxidation or reduction of a dye. This allows for both qualitative and quantitative results to be measured, or for the storage of information in the case of memory units [48].

Conclusions and Future Perspectives

With small molecule gene silencing strategies on the rise, DNAzymes have great potential as therapeutic agents. The apparent lack of off-target effects commonly seen in higher organisms for gene silencing approaches using antisense and siRNA, is possibly a result of the self-sufficient cleavage properties of DNAzymes, whereas siRNAs operate via the cell endogenous Dicer/RISC mechanism [51]. Sequence composition induced interferon responses common to antisense RNA mediated long double-stranded RNA molecules, which cause a more global mRNA degeneration in mammals [52] have not been described for DNAzymes so far. Nevertheless, similar to any establishment of siRNA mediated gene knockdown, a range of molecules might need to be tested to achieve optimal target RNA cleavage and control experiments using a randomized sequence flanking the catalytic core instead of the target binding sequence are imperative. Toxic effects due to the use of cationic lipofectants as DNAzyme delivery method have been described [53]. Since DNAzymes act through selective binding and cleavage of mRNA, this method, like all gene silencing approaches will merely achieve a knockdown of mRNA transcripts. Therefore unlike classic and conditional gene targeting by homologous recombination [54] DNAzymes might be limited in modeling gene function and disease, depending on the relevance of the target gene or when a complete loss-of function approach is crucial. The potential risk of DNA mediated mutagenesis due to random integration of foreign DNA fragments into the host genome is further to be considered when using DNAzymes as therapeutic agent. While DNA is generally more stable than ribonucleic acids, DNAzymes, too, are vulnerable to exonuclease-mediated degradation, and might therefore require direct delivery to a target cell. This could exclude DNAzymes from the treatment of pathophysiological conditions where bioavailability is challenging due to difficulties in DNAzyme delivery or impaired cellular uptake thereof. Similar to work with antisense oligonucleotides, ribozymes and aptamers, modifications to increase DNAzyme half-life for in vivo applications have been explored [53,55-58]. Phosphorothioate modifications to confer endonuclease resistance as commonly used in ribozymes and aptamers inflict a similar toxic and off-target effect when used in DNAzymes [59-62]. Other modifications, like terminal stem-loop hairpins of the substrate binding arms [63] or a 3’-3’ inverted nucleotide at the 3’terminus of the DNAzyme molecule [57] appear promising.

With the continued development of advanced artificial biomolecular motifs, such as DNAzymes, bio-computational and biomedical architectures, solutions previously only envisioned in science fiction writings, become more realistic. The potential for bio-catalytic species, like traditional protein enzymes, produced from highly specific and robust components, like DNA, is limitless. One obvious field that could be significantly impacted is that of biofuel cell technologies. Typically constructed from redox enzymes or nanoparticles, biofuel cells utilize their catalytic nature to extract energy from their surroundings [43]. One of the most potentially hopeful of these circumstances is the implantation in animals and humans, where glucose and oxygen are typically used as “fuel”. The goal is for this harvested energy to be used to power implanted electronics, such as pacemakers [64]. One of the greatest limitations facing modern implantable biofuel cells is the long-term stability of the enzymes immobilized on their surface. If DNAzymes were developed that were capable of the bio-catalytic redox catalysis of glucose and oxygen, the most common sources of fuel for implantable biofuel cells [43], future biofuel cell electrodes could be developed with the ability to outlast even the most robust protein-based enzyme electrodes.

Future applications for DNAzymes in cancer treatment are apparent, and steps have been taken towards their realization. Continued research and interest in this field could result in a highly specific and effective DNAzyme-based anti-tumor cell treatment. Given the high specificity of DNAzymes, treatments could be tailored to specific mutations of a cancer cell; for example by targeting specific nucleotide sequences that result in cancerous characteristics and therefore distinguish the cancer cell from healthy neighbors. Additionally, being composed of bare DNA, DNAzymes inherently represent a minimal risk of unintended toxicity, unlike current chemotherapy compounds. This being said, any technology involving the direct interaction with a host cell’s nucleic acids must be carefully tested and regulated to ensure its safety by minimizing off-target effects. No cancer treatment would be beneficial to any patient if the induction of apoptosis would not be tumor cell-specific.

DNAzymes show promising potential in many other areas aside from the ones mentioned above. Whenever targeting at a molecular level is required, DNAzymes might be of use, for example in targeting RNAs encoding viral structures causative of human immunodeficiency virus infection (HIV) [65], dengue fever [66], “bird flu” (H1N1 and related strains) [67], severe acute respiratory syndrome (SARS) [68] and more recent threats to society like the middle east respiratory syndrome (MERS) or Ebola. DNAzymes could provide a novel option in such areas desperate for urgent solutions.

Treatment with DNAzyme DZ1 targeting the late membrane protein 1 (LPM1) of the Epstein Barr virus, an oncogenic factor thought as major contibutor in nasopharyngeal carcinoma (NPC) showed increased radiosensitivity and decreased angiogenesis in LPM1-induced NPC [69]. Another clinical trial to test the efficacy and safety of the DNAzyme SB010 in allergen-induced asthmatic responses aiming at silencing GATA3 was also recently completed [70] , while trials for a topical application of SB011 to treat mild to moderate atopic dermatitis as well as intrarectally applied SB012 to treat active ulcerative colitis are currently recruiting (www.ClinicalTrials.gov). Technological advances allow for further fine tuning of the activity of DNAzymes by using a L-ribose (L-RNA) sugar backbone instead of the naturally occurring D-ribose (D-RNA) [71,72]. This has the potential to overcome current obstacles for broader implications of DNAzymes in bioanalytical, therapeutic or diagnostic applications hampered by RNAse-mediated substrate degradation in complex biological samples [72]. In combination with a low immunogenicity of DNAzymes and high precision at a nucleic acid base level, DNAzymes have the potential to excel in personalized medicine just as much as in biofuel cell technologies. Future combined efforts of biomolecular, biochemical and bioinformatic sciences improving DNAzymes and identifying more possible targets and applications will also impact on the evolving field of Synthetic Biology [6,73] (see Figure 1).

Figure 1: Multidisciplinary efforts make way for current and future applications for DNA Enzyme Systems in the medical and biotechnological fields. (DNAzyme schematic adapted from [74])

Acknowledgements:

This review was inspired by course BY586, a trans-disciplinary upper level course on Molecular Biotechnology at Clarkson University.

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