Invest New Drugs DOI 10.1007/s10637-015-0213-y

REVIEW

Aptamers as a novel tool for diagnostics and therapy Onat Kadioglu & Anna Helena Malczyk & Henry Johannes Greten & Thomas Efferth

Received: 8 January 2015 / Accepted: 21 January 2015 # Springer Science+Business Media New York 2015

Summary Aptamers are short single-stranded DNA or RNA oligonucleotides that are capable of binding small molecules, proteins, or nucleotides with high specificity. They show a stable conformation and high binding affinity for their target molecules. There are numerous applications for aptamers in biotechnology, molecular diagnostics and targeted therapy of diseases. Their production is cheap, and they generally display lower immunogenicity than monoclonal antibodies. In the present review, we give an introduction to the preparation of aptamers and provide examples for their use in biotechnology, diagnostics and therapy of diseases. Keywords SELEX . Targeted therapy . Biosensor . Molecular diagnostics . Peptides

Introduction Aptamers are short single-stranded DNA or RNA oligonucleotides that are capable of specifically binding small chemical molecules, proteins, or nucleotides. The name aptamer refers to the Latin word aptus meaning attached, tied, tethered. They reveal a stable conformation and high binding affinity for their target molecules. Like antibodies, this class of molecules work according to the lock-and-key principle, which makes them O. Kadioglu : A. H. Malczyk : T. Efferth (*) Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany e-mail: [email protected] H. J. Greten Biomedical Sciences Institute Abel Salazar, University of Porto, Porto, Portugal H. J. Greten Heidelberg School of Chinese Medicine, Heidelberg, Germany

suitable for the detection and deactivation of specific molecules. Chemically synthesized aptamers are produced using an in vitro selection technique referred as systematic evolution of ligands by exponential enrichment (SELEX). In this method, high sequence specificity and binding properties of aptamers are identified from a mixture of oligonucleotides. In comparison, to antibodies, aptamers reveal a number of advantages. Their preparation is relatively easy and inexpensive, and they reveal lower immunogenicity than antibodies, but many have better specificity, higher affinity to their target, and less non-specific cross-reactivity than antibodies [1–5]. Antibodies bind to larger molecules, but aptamers bind to a larger range of structures such as proteins, cells, or small molecules (organic dyes, amino acids, metal ions, nucleotides) [6]. Aptamers can be used to detect toxic compounds or even non-immunogenic targets [6]. Furthermore, aptamers are heatand pH stable and are more resistant to organic solvents than antibodies [7–9]. Unlike antibodies, aptamers can be denatured and renatured multiple times without loss of activity [10]. Aptamers are extremely versatile to produce chimeric aptamers, such as coupling of two aptamers or coupling of aptamers to nanoparticles [11]. Furthermore, aptamers can be linked to ribozymes. These constructs are termed aptazymes and represent combinations of ligand-binding domains (aptamers) and ribozymes to knock-out gene expression [12–15]. Moreover, aptamers can chemically be coupled to siRNAs, fluorophores, radioisotopes, electrochemical or Raman reporters, functional groups at 5’ or 3’ end or on the DNA backbone or on bases making them exquisitely suitable as carrier molecules both for diagnosis and therapy [16, 17]. A disadvantage of aptamers is their short half-life time in blood which may be due to their small size, thus promoting renal clearance and nuclease degradation [18]. Several strategies have been developed to prevent rapid degradation, e.g., their conjugation with polyethylene glycol to increase in vivo activity [19] or modifications with aminopyridine, 2-

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fluoropyrimidine or 2’-O-methyl nucleosides [10, 20–23]. It has also been suggested to use spiegelmers, which are resistant to nuclease degradation [24, 25], as spiegelmers consist of Lnucleotides and nucleases recognize only D-nucleotides. The first chemically synthesized therapeutic aptamer, Pegaptamib, was approved in December 2004 by the U.S. Food and Drug Administration (FDA) for the treatment of ″ age-related macular degeneration″ (AMD). It covalently binds to vascular endothelial growth factor (VEGF) in the eye, which is responsible for pathological neovascularization and blood vessel permeabilization. Aptamers are also promising for future diagnostic and therapeutic use in cardiovascular, malignant and ophthalmological diseases as well as for biological and medical research [26–28]. In the present review, we focus on the production and use of aptamers in biomedical research and clinical applications with special emphasis in oncology.

Definition and properties of aptamers The term aptamer has been coined by Ellington and Szostak [29] and comes from the Latin word ″aptus″ and the Greek word ″meros″, which can be translated as ″fitting to a region ″[26, 28]. Aptamers are single or partially double-stranded DNA or RNA oligonucleotides, 15–70 nucleotides in length that attach with high affinity to specific regions of their target molecules. The three-dimensional folding of the aptamers allow highly specific binding by hydrogen bonding, hydrophobic, coulombic and van der Waals interactions between the aptamer and the target molecule. Aptamer binding affinities are generally between 1 pM and 1 μM and binding affinities between aptamers and proteins are commonly in the nanomolar range. This is equivalent to the binding affinities of monoclonal antibodies. Target molecules of aptamers include small molecules such as metal ions, dyes, and theophylline; nucleotides (e.g., ATP); peptides and proteins (e.g., thrombin); glycoproteins (e.g., CD4); and antibodies. In fact, aptamers that can bind membrane fragments or entire cells have been isolated [3, 26, 27]. Similar to antibodies, aptamers can be used for the detection and sometimes the functional inhibition of other molecules. In contrast to monoclonal antibodies, aptamers are chemically synthesized and can bind under nonphysiological conditions to their target molecules. They reveal lower immunogenicity than antibodies and may have a higher specificity and binding affinity to a given molecule. Examples are the chemically related molecules theophylline and caffeine, which can be distinguished by means of aptamers better than by antibodies [3]. Furthermore, aptamers can be synthesized to bind poorly immunogenic or toxic substances [3, 27]. In contrast to monoclonal antibodies, nucleotides are digested by DNA and RNA nucleases within seconds to

minutes in biological fluids. Therefore, aptamers must be resistant to digestion for use in vivo. This can be achieved in one of two ways: Either nuclease-modified nucleotides can be incorporated into the RNA pool before SELEX selection is performed, or unmodified nucleotides can be subsequently replaced by modified ones [27]. Another disadvantage of aptamers is that the variety of three-dimensional structures which oligonucleotides can generate is not as large as of peptides or proteins. One subgroup of aptamers is the so-called spiegelmers, which are L-enantiomeric RNA or DNA oligonucleotides. These molecules display a higher resistance to enzymatic degradation than D-oligonucleotides due to their unnatural mirror image chirality [30].

Preparation of nucleic acid aptamers For production aptamers by SELEX, a randomized oligonucleotide library generated by solid-phase synthesis is screened via a target molecule binding event or catalytic process to isolate aptamers with the desired characteristics [26]. A library of randomized RNA or DNA oligonucleotides that differ in their three-dimensional structures and binding affinities is used to begin the SELEX process. The specific structures and binding affinities of aptamers in the library arises from the distinct sequences of nucleotides. In several selection cycles, this oligonucleotide library is incubated with the target molecule. Those oligonucleotides with the highest affinity for the target molecule are positively selected and amplified [27] (Fig. 1). The chemical synthesis of aptamers The oligonucleotides are synthesized by solid-phase synthesis, in which a nucleoside covalently binds to a polymeric carrier. The resulting nucleotide chain is extended by another nucleoside in a mobile phase, which binds to the first nucleoside’s free 5′-OH group. A protecting group attached to the 5′OH of the free nucleosides is used to prevent multiple binding events so that the length of the aptamer can be controlled. Subsequently, the non-binding components are washed from the carrier on the mobile phase and the reaction product remains immobilized to a carrier [32]. The protecting group is then removed from the bound nucleoside so that the next base can be added. Mesitylenesulfonyl-3-nitro-1,2,3-triazole (MSNT) is used as a condensation reagent for the binding of nucleosides to the growing chain. In vitro selection Most DNA and RNA libraries contain 30–60 nucleotide-long randomized sequences, which are produced by random

Invest New Drugs Fig. 1 In vitro selection of aptamers by SELEX. Oligonucleotides of a randomized library are subjected to selection. A target molecule binds oligonucleotides, unbound oligonucleotides are washed out. After washing, the bound oligonucleotides are separated again from the target molecule and amplified. Random or targeted mutants are generated during the amplification step, and join the population of aptamers that are selected in further SELEX cycles. After the last cycle, cloning and sequencing of isolated aptamers are performed [31]

Cloning and sequencing

Nucleotide library

Targets Characterization and research

EVOLUTION Mutagenesis during amplification Removal of binding nucleotides

SELEX 6-20 cycles

SELECTION e.g. affinity chromatography

Removal of non-binding nucleotides

Regeneration of targets

nucleotide incorporation during chemical synthesis. For technical reasons however, the synthesis rarely exceeds 1015–1016 different molecules. The aptamers are flanked at the 3 ′and 5′ ends by 15–20 nucleotide primer sequences, which are necessary for amplification in polymerase chain reaction (PCR) and for in vitro transcription (for RNA selection). The forward primer sequence is located at the 5′-end and the reverse primer at the 3 ′OH of the randomized region [26, 27]. Selection of the binding nucleotides is usually performed by affinity chromatography. Molecules with high affinity bind to pre-immobilized or immobilized target molecules. The complexes of oligonucleotides and the target molecules differ in their stability depending on their affinity for the target molecule. Target molecules and bound nucleotides remain on a selection matrix or filter, and can be separated from the weakly bound or non-bound molecules by washing with a liquid phase. The binding molecules are then extracted and are available for further selection steps. DNA oligonucleotides are PCR-amplified. RNA oligonucleotide aptamers are reversed transcribed into DNA before amplification and then subjected to selection. All enzymatic steps of SELEX, especially the PCR amplification using Taq polymerase, are error-prone and thus can also be used for mutagenesis. Each amplification step results in modified oligonucleotides. Hence, SELEX represents an evolutionary process. The affinity of the oligonucleotides for the target molecules stops to increase after 6–20 cycles. At this time, the library of isolated aptamers is cloned and sequenced, in order to preserve individual sequences and to determine the base sequence of high-affinity aptamer oligonucleotides. SELEX is a relatively simple technology to produce and isolate molecules that are directed against a target protein. However, aptamers must be made resistant to nucleases if they

are to be used in biological fluids. For this purpose, chemically-modified nuclease-resistant libraries are available for use in vitro selection. In many cases, post-SELEX incorporation of nuclease-resistant nucleotides is difficult to achieve, since subsequent modifications are usually accompanied by a loss of affinity. To create nuclease-resistant aptamers, the use of 2′fluoropyrimidine triphosphates and 2′-amino-pyrimidine triphosphates has been successful. These nucleotides are degradation-resistant and are tolerated as components of the template strand by both RNA polymerases and reverse transcriptases, and thus can be used for pre-SELEX modification. Aptamers derived from 2′ -amino-modified RNA pyrimidinetriphosphate library reveal a 300–1,000-fold higher stability than unmodified RNA oligonucleotides. Another possibility to stabilize the aptamers is the exchange of 2′-hydroxy-purine nucleotides with 2′-O-methyl groups. However, the exchange must be performed slowly and gradually after selection (post-SELEX), since the effect on the aptamer-binding properties is hardly predictable. The reversal of the selected oligonucleotides in their L-enantiomeric forms may further increase the stability. The resulting so-called spiegelmers reveal the same target affinity, but a lower susceptibility to enzymatic degradation. An example for a stable aptamer is pegaptamib (Magugen®), which is 2′-fluoro-modified at all pyrimidines and 2′-O-methyl-modified at all purines. Additionally, there is a polyethylene glycol residue at the 5′ end and an inverted nucleotide at the 3′-OH end [26, 27]. Automated in vitro selection After Ellington and Stostak developed the manual in vitro selection method [29], an automated 96-well microtiter

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plate-based technique was introduced. Target molecules are immobilized on magnetic particles, permitting the binding oligonucleotides to be separated by a magnet. The automated process is monitored via PCR products, which are analyzed after each amplification step. The robotic workstation is connected to a vacuum chamber in which aptamers can be selected using free, nonimmobilized target molecules (e.g., proteins). The vacuum allows for separation of target molecules and their bound oligonucleotides from non-binding nucleotides using a filter. Further improvements of the selection process have been aimed at the simultaneous selection of target molecules bound to different oligonucleotides. Another automated selection process developed by Eulberg et al. [33] makes it possible to directly monitor the selection course and to identify binding oligonucleotides. Here, binding reactions to the target molecule with different affinities take place in parallel. A non-cross contamination (NCC) system and amplification and purification of the in vitro transcription products represent further novel developments. Crosscontamination is prevented by the NCC system covering each well of the 96-well plate with a lid that can be individually opened or closed with a lid opener. Aliquots from concomitant PCR reactions are taken, the double stranded DNA is stained with the fluorescent dye PicoGreen, and the amount of amplification products is measured in order to investigate the enrichment of sequences in the selection process. The oligonucleotides are amplified in subsequent cycles, if a predetermined threshold is not reached. The oligonucleotides are washed on filter units following the selection process [27].

Fields of application Aptamers can be applied both in diagnostics and therapy. In fact, some aptamers are already in preclinical or clinical trials [3]. Although monoclonal antibodies are still more widespread than nucleic acid-based products as therapeutic agents, oligonucleotides are becoming increasingly important. There are many different therapeutic functions of oligonucleotides. For example, some sequence-specifically hybridize to RNA and Bsilence^ its translation. Others bind to target molecules and inhibit them or provoke immunostimulatory effects, as is the case with CpG DNA [27]. The binding of natural oligonucleotides to proteins and the resulting change in the protein’s mechanism of action has been identified in the interaction of viral molecules with host proteins. Such discoveries have raised interest in these molecules for therapeutic purposes, especially since their lack of immunogenicity is a great advantage [28]. In the following section, some examples of various aptamer applications are described.

Aptamers in biotechnology and toxicology Aptamers may be used in cell biology to inhibit cellular signaling pathways. They may also be applied in biotechnology to develop specific biomarkers and molecular sensors [34]. In diagnostics, aptamers may detect toxic substances or diseaserelated molecules [35, 36]. Reprogramming of bacteria by aptamers Another possible application of aptamers in biotechnology is for reprogramming of bacteria. For example, Joy Sinha and colleagues used aptamers to reprogram bacteria to identify the herbicide atrazine in a mixture of chemicals and to specifically destroy it. Atrazine is commonly used in the U.S., but is banned in Europe. Its activity is based on binding of quinone QB and blockade of electron transport in photo-system II of photosynthesis. As a persistent environmental pollutant, atrazine is toxic to both plants and animals [35, 37]. Atrazine can be converted into the non-toxic hydroatrazine by the enzyme chlorohydrolase in animals and plants. Sinha et al. [35] identified a riboswitch able to induce protein translation in the presence of atrazine. As a next step, the bacteria were reprogrammed in such a way that they revealed increased motility in the presence of atrazine and catalyzed the conversion of atrazine to hydroatrazine. Oligonucleotides binding to atrazine have been identified by SELEX. To isolate an appropriate riboswitch, aptamers carrying a random sequence and a so-called RNA cheZ sequence, which is known to mediate motility in Escherichia coli (E. coli), were synthesized via molecular cloning. The positively selected aptamers were inserted into CheZ-deficient E. coli, which were subsequently seeded in semi-solid medium. Bacterial colonies that were immobile in the absence and mobile in the presence of atrazine were selected. Using a β-galactosidase assay, the riboswitches were then incorporated into a lacZ construct. For conversion of the toxic atrazine into the non-toxic hydroatrazine, the atzA gene encoding the atrazine chlorohydratase was inserted. The in vivo study showed that the ribsoswitch combining cheZ for atrazine-induced mobility increase and atzA for atrazine metabolism of led to detoxification of the herbicide [35]. Aptamer-based short-term test for cocaine In 2006, an aptamer-based test for cocaine detection in body fluids was developed by Yi Lu and co-workers from the University of Illinois (Urbana, USA) [34]. This assay is reliable, and is much easier to perform than standard laboratory methods. The test involves adding the sample (e.g., saliva, urine or blood serum) to a test strip. If a red-stained band appears, the sample contains cocaine.

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The test is based on a mixture of gold spheres and aptamers that bind cocaine. The test strip is put into the sample, and the liquid migrates along the strip to small lumps of nanoscopic gold beads and aptamers. Complementary DNA aptamer sequence and biotin are bound to the gold beads. The DNA sequences hybridize forming gold bead aggregates. Upon contact with cocaine, the aggregate dissolves as the DNA aptamers bind to cocaine. The liquid with gold bead-aptamer aggregates and also unbound gold spheres migrates to a membrane. Only unbound beads can pass through the membrane; as aggregates are too large to pass. The unbound beads are transported to a streptavidin-strip and the biotin-labeled gold spheres bind to the streptavidin in order to concentrate them in the strip. The presence of the gold beads in this region of the test strip results in a red color. It is conceivable that this method can also be applied for shortterm tests to detect other drugs, toxins, and environmental pollutants [34]. Aptamers for diagnostics Aptamers may be applied for diagnostics in cardiology, ophthalmology or oncology. As an example, Ilyas et al. [36] took advantage of aptamers for the electrical detection of cancer cells. They used an aptamer that was able to bind to the epidermal growth factor receptor (EGFR), a surface protein that is over-expressed in many cancer types and is one of the most frequently occurring oncogenes. The EGFR concentration in blood serum is elevated in cancer patients and represents a measure of the stage of disease. Even small concentration changes could be critical for disease progression and treatment failure. Therefore, it is particularly important to develop an efficient and highly sensitive detection technique. Ilyas and colleagues generated Bnanogaps^ between gold electrode pairs by scratching the surface with a focused ion beam. These nanogaps produced less than one nanometer wide disruptions between the gold electrodes. The aptamer and a DNA sequence complementary to the aptamer are bound to a gold electrode on a SiO2 surface. The current increases by up two orders of magnitude upon binding of EGFR to the aptamer depending on the EGFR concentration. This biosensor technique is cheaper and more sensitive than other methods of EGFR determination and, hence, holds great potential for the development of diagnostic applications in the future [36]. Aptamers as drugs Another application of aptamers is in the treatment of diseases. Macugen® is the first clinically approved aptamer and more aptamers are in the drug development pipeline.

Ophthalmology Macugen® contains the aptamer pegaptanib and was FDA and EMEA approved in 2004 and 2006, respectively. This 28 nucleotide long RNA aptamer is modified at the 5′-end with a 40 kDa polyethylene glycol residue to prevent degradation by nucleases and to prolong the drug’s half-life. Macugen® is a drug to treat choroidal neovascularization (CNV), which causes accelerated loss of vision in age-related macular degeneration (AMD) [38]. Neovascularization is caused by increasing the concentration of the vascular endothelial growth factor (VEGF). The mechanism of pegaptanib is dependent on binding between VEGF and the aptamer [39]. This binding prevents VEGF from binding to the VEGF receptor and activating downstream signaling pathways [40–42]. Pegaptanib binds to the A164/165-isoform of VEGF with high affinity, but only with low affinity to the A120/121-isoform. Therefore, this aptamer has the advantage that other important physiological functions of VEGFs are not significantly influenced. For instance, the aptamer is capable of effectively inhibiting leukocyte adhesion and vascular growth, which both take place during the neovascularization processes, while the physiological VEGF-regulated vessel maturation is not affected. After insertion of a polyethylene glycol residue, the half-life of pegaptinib was 9.3 and 20 hours for intravenous or subcutaneous injections respectively in Rhesus monkeys. Pegaptanib was stable in human blood plasma for more than 18 hours. As recently shown in a clinical trial, pegabtanib can be successfully used against neovascular AMD and side effects such as endophthalmitis, a serious intraocular inflammatory disorder, or retinal detachment were rare. Common side effects of other VEGF inhibitors, such as high blood pressure, bleeding, and thromboembolism, were not observed with pegabtinib [43]. However, although the efficacy of the drug against neovascularization has been demonstrated, a significant slowing of vision loss in diseased patients was not observed. Oncology Aptamers are also promising for use in cancer treatment. They can bind to components of signaling pathways and disrupt signaling, antagonistically bind to receptors, activate immune responses or target adherence factors [44, 45]. The prostate-specific membrane antigen (PMSA) has been used by several authors as target to inhibit prostate cancer cells. The prostate-specific membrane antigen (PMSA) has been used by several authors as target to inhibit prostate cancer cells. Lupold et al. [46] developed nucleasestabilized RNA aptamers and Chu et al. [47] aptamers coupled to the cytotoxic compound gelonin to specifically kill prostate tumor cells.

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Stecker et al. [48] showed that an aptamer-C1q-conjugate coupled to the classical complement system was able to selectively kill cancer cells. This aptamer bound the MUC1 protein on the plasma membrane of human breast adenocarcinoma cells (MCF7). In turn, binding of the biotinylated aptamer led to initiation of the classical complement system when the biotin bound to a streptavidin-conjugated C1q. Förster resonance energy transfer measurements (FRET) showed that membrane depolarization occurred upon aptamer binding, suggesting the formation of a membrane-attack complex (MAC). As demonstrated by transmission electron microscopy and immunogold labeling, aptamer-mediated complement-fixation process forms a MAC. MAC-induced osmotic cell swelling may then lead to cell death. This experiment shows that not only monoclonal antibodies, but also much cheaper aptamers can initiate the immune system and kill cancer cells [48]. Another approach was introduced by Liu et al. [49], who examined possibility of improving the transport of the anticancer drug doxorubicin to HER2-positive breast cancer cells using aptamers. HER2 is an oncoprotein that is overexpressed in a variety of malignant tumors such as breast, ovarian and lung cancer, and is a frequent target for tumor-specific delivery of therapeutic agents. The authors coupled doxorubicin to a DNA aptamer, HB5, which binds to the extracellular domain of HER2. The HB5-doxorubicin complex thus specifically targeted HER2-positive breast cancer cells, while HER2negative cells were not affected, resulting in reduced toxicity of the chemotherapy. Thus, aptamers alone or coupled to anticancer drugs open a wide range of novel therapeutic possibilities. Aptamers hold great potential as a new principle for therapy and for the development of more efficient and specific treatment options with less severe side effects. Cao et al. [50] developed a liposome with DNA aptamerfunctionalized cisplatin to target nucleolin and kill cancer cells. An LNA-aptamer complex targeting both nucleolin and EpCAM loaded with superparamagnetic iron oxide nanoparticles was able to distinguish between tumor and normal cells [51]. The absorption of iron in growing cells is increased and there is a relationship between iron uptake and the rate of tumor proliferation [52]. This explains higher inhibition rates of the LNA-aptamer nanoparticle in tumor cells than in normal cells. An interesting approach has been described by McNamara et al. [53]. The authors presented multivalent aptamers that bind the 4-1 BB protein and co-stimulate CD8+ T-cells to inhibit tumor growth in vivo. Other diseases The application of aptamers is getting more and more attractive as a new treatment principle. In addition to the potential use of aptamers in ophthalmology and oncology,

aptamers have been proposed as new treatments for a number of other diseases. Aptamers have been designed to inhibit blood clotting by targeting coagulation factors or thrombin and prothrombin making them useful for anticoagulant therapy [54–56]. A wide field of therapeutic applications are viral and other infectious diseases. Several investigators reported the inhibition of hepatitis C virus (HCV) by RNA aptamers that target the HCV NS3 helicase domain [57–61] or HCV internal ribosome entry site (IRES) [62, 63]. DNA aptamers binding the RNA-dependent RNA polymerase of HCV have been also found to inhibit viral RNA synthesis [64]. Human immunodeficiency virus 1 (HIV-1) has been inactivated by inhibiting the HIV-1 reverse transcriptase, HIV-protease or HIV integrase [65–67]. Zhou et al. [68] described dual inhibitory aptamers that have been coupled to siRNAs as novel delivery systems for HIV-1 therapy. RNA aptamers have also been applied to block infections caused by the human cytomegalovirus or influenza virus [69]. Neurodegenerative diseases such as Alzheimer’s Disease can be approached as well as shown by aptamers that target the Abeta peptide [70].

Conclusion and perspectives Aptamers may be used in various fields of biotechnology, molecular diagnostics, and targeted therapy of diseases. Their production is cheap, and they have the advantage of lower immunogenicity as compared to monoclonal antibodies. The disadvantage of aptamers, however, is their relatively low half-life in biological systems, which necessitates chemical modification to reduce degradation. Furthermore, the SELEX technology requires some experience to select suitable aptamers from an oligonucleotide library [3]. Aptamers can be used for diagnostics, basic research and therapy of various diseases, including cancer. The fact that the number of publications on aptamers has multiplied in recent years demonstrates their enormous potential as alternatives to proteins and monoclonal antibodies in biological and biomedical applications [3]. Conflict of interest The authors declare that they have no conflict of interest.

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