**1.3 Aptamer production: selection and identification of aptamers**

Aptamers are a new class of recognizing agents that came into light in 1990 [16]. Tuerk and Gold developed a method known as systematic evolution of ligands by exponential enrichment or popularly called as SELEX method for selection of aptamers against target [17]. The method is in vitro in nature and does not require the use of animals. Aptamers are chemically synthesized and selected for their high affinity and specificity for a certain target through the SELEX process (**Figure 2**).

Since the early 1990s, systematic evolution of ligands by exponential enrichment

and similar methods have been reported to efficiently select RNA and DNA aptamers [10]. Thereafter, nucleic acid aptamers have been extensively researched and applied. The identification of aptamers is achieved by an in vitro selection process, also termed SELEX. In this method, a synthetic nucleic acid library that contains up to 1015 different sequences and structures is incubated with the desired target molecule, unbound sequences are removed, and target-linked sequences are recovered and amplified using PCR or RT-PCR [3, 18]. This cyclic process is repeated several times (5–16) until the nucleic acid population has been substantially enriched for target-specific sequences. Nucleic acid libraries offer the largest collection of compounds available so far for screening and selection purposes. The monoclonal aptamer sequences are accessible by cloning and sequencing these libraries. But the latest generation sequencing approaches allow in-depth evaluation of the selection process [19, 20]. A wide range of selection methods and strategies have been described since the first reports in 1990. The basis of these variations is based on the separation method used or chemical modifications added to the nucleic

*Aptamers and Possible Effects on Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89621*

**2. The application of aptamers as molecular tools in neurosciences**

nal cell behavior by inhibiting specific proteins.

on BACE1 activity in an AD cell model [23].

**277**

Aptamers have a number of diagnostic and therapeutic applications, such as biosensors and target inhibitors. Due to simple preparation, easy modification, and stability, aptamers have been used in the diverse areas within molecular biology, biotechnology, and biomedicine [2]. In general, aptamers are used in diagnostics, pathogen recognition, cancer recognition, stem cell recognition, monitoring environmental contamination, biosensors, and therapeutics. Aptamers are being used in versatile applications. However, their use as a molecular research tool in neuroscience is limited. There are very few studies on the production of aptamers targeting neuroscience-related target proteins such as ion channels or application for neuro-

Neurodegenerative diseases are defined as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. Alzheimer's disease (AD) is the most common cause of dementia and is characterized by progressive loss of memory and other cognitive functions. It is considered a major epidemic worldwide, where currently more than 35 million people live with this disease. By 2050 it is estimated this figure will reach 115 million [21]. AD is characterized by two major abnormalities; abnormal extracellular amyloid β-protein (Aβ) disposition and intracellular neurofibrillary tangle (NFT) formation, both leading to neuronal degeneration. The generation of Aβ is triggered by B-site amyloid precursor protein cleaving enzyme 1 (BACE1). Thus, BACE1 is a prospective target for interfering with Aβ production and the treatment of AD [22]. A DNA aptamer selected by Liang et al. has been shown to bind to BACE1 with high affinity and good specificity, exhibiting a distinct inhibitory effect

Autoimmunity and autoantibodies play a role in the pathogenesis of many diseases. Recently, a research team in Germany demonstrated the presence of functional autoantibodies against G-protein bound receptors in the serum of Alzheimer's and vascular dementia patients [24]. And the aptamer BC007, which is selective for the GPCR-AAB FAB fragment as a "broad-spectrum neutralizer," has been developed based on binding and epitope mapping studies [24]. The fact that BC007

acid libraries.

The process starts with synthesizing a random DNA oligonucleotide library (**Figure 2**). This library consists of a diverse pool of ssDNA fragments (1015). When selecting RNA aptamers, the library needs to be converted into an RNA library. Subsequently, the target is introduced in the pool, non-binding fragments are removed by several washing steps, and the remaining fragments are amplified by PCR or RT-PCR. A new pool of oligonucleotides is created using the selected fragments and another round is performed. Usually 8–15 rounds are performed in order to obtain a high-affinity aptamer.

**Figure 2.** *Schematic illustration of an in vitro selection cycle.*

*Aptamers and Possible Effects on Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89621*

have been recognized as therapeutic molecules due to their anticancer and

Aptamers are a new class of recognizing agents that came into light in 1990 [16]. Tuerk and Gold developed a method known as systematic evolution of ligands by exponential enrichment or popularly called as SELEX method for selection of aptamers against target [17]. The method is in vitro in nature and does not require the use of animals. Aptamers are chemically synthesized and selected for their high affinity and specificity for a certain target through the SELEX process (**Figure 2**). The process starts with synthesizing a random DNA oligonucleotide library (**Figure 2**). This library consists of a diverse pool of ssDNA fragments (1015). When selecting RNA aptamers, the library needs to be converted into an RNA library. Subsequently, the target is introduced in the pool, non-binding fragments are removed by several washing steps, and the remaining fragments are amplified by PCR or RT-PCR. A new pool of oligonucleotides is created using the selected fragments and another round is performed. Usually 8–15 rounds are performed in

**1.3 Aptamer production: selection and identification of aptamers**

antivirus activities.

**Figure 2.**

**276**

*Schematic illustration of an in vitro selection cycle.*

order to obtain a high-affinity aptamer.

*Neuroprotection - New Approaches and Prospects*

Since the early 1990s, systematic evolution of ligands by exponential enrichment and similar methods have been reported to efficiently select RNA and DNA aptamers [10]. Thereafter, nucleic acid aptamers have been extensively researched and applied. The identification of aptamers is achieved by an in vitro selection process, also termed SELEX. In this method, a synthetic nucleic acid library that contains up to 1015 different sequences and structures is incubated with the desired target molecule, unbound sequences are removed, and target-linked sequences are recovered and amplified using PCR or RT-PCR [3, 18]. This cyclic process is repeated several times (5–16) until the nucleic acid population has been substantially enriched for target-specific sequences. Nucleic acid libraries offer the largest collection of compounds available so far for screening and selection purposes. The monoclonal aptamer sequences are accessible by cloning and sequencing these libraries. But the latest generation sequencing approaches allow in-depth evaluation of the selection process [19, 20]. A wide range of selection methods and strategies have been described since the first reports in 1990. The basis of these variations is based on the separation method used or chemical modifications added to the nucleic acid libraries.

#### **2. The application of aptamers as molecular tools in neurosciences**

Aptamers have a number of diagnostic and therapeutic applications, such as biosensors and target inhibitors. Due to simple preparation, easy modification, and stability, aptamers have been used in the diverse areas within molecular biology, biotechnology, and biomedicine [2]. In general, aptamers are used in diagnostics, pathogen recognition, cancer recognition, stem cell recognition, monitoring environmental contamination, biosensors, and therapeutics. Aptamers are being used in versatile applications. However, their use as a molecular research tool in neuroscience is limited. There are very few studies on the production of aptamers targeting neuroscience-related target proteins such as ion channels or application for neuronal cell behavior by inhibiting specific proteins.

Neurodegenerative diseases are defined as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. Alzheimer's disease (AD) is the most common cause of dementia and is characterized by progressive loss of memory and other cognitive functions. It is considered a major epidemic worldwide, where currently more than 35 million people live with this disease. By 2050 it is estimated this figure will reach 115 million [21]. AD is characterized by two major abnormalities; abnormal extracellular amyloid β-protein (Aβ) disposition and intracellular neurofibrillary tangle (NFT) formation, both leading to neuronal degeneration. The generation of Aβ is triggered by B-site amyloid precursor protein cleaving enzyme 1 (BACE1). Thus, BACE1 is a prospective target for interfering with Aβ production and the treatment of AD [22]. A DNA aptamer selected by Liang et al. has been shown to bind to BACE1 with high affinity and good specificity, exhibiting a distinct inhibitory effect on BACE1 activity in an AD cell model [23].

Autoimmunity and autoantibodies play a role in the pathogenesis of many diseases. Recently, a research team in Germany demonstrated the presence of functional autoantibodies against G-protein bound receptors in the serum of Alzheimer's and vascular dementia patients [24]. And the aptamer BC007, which is selective for the GPCR-AAB FAB fragment as a "broad-spectrum neutralizer," has been developed based on binding and epitope mapping studies [24]. The fact that BC007

aptamer was successful in neutralization in an in vitro study revealed the possibility of being a potential therapeutic tool for dementia patients.

Parkinson's disease (PD), the second most common neurodegenerative disease after AD, affects over 7 million people worldwide. The pathology is characterized by loss of dopaminergic neurons, leading to decreased production of dopamine, a neurotransmitter that regulates movement and cognition. In the previous immunotherapy, targeting the α-syn in PD models with monoclonal antibodies has established α-syn protein as an effective target for neuronal cell death. The pathogenesis of Parkinson's disease involves the accumulation of α-synuclein protein in neurons. Anti-α-synuclein antibody treatment has achieved some success. However, this antibody-based immunotherapy is limited by the inherent immunogenicity of antibodies and the inability of antibodies to reach intracellular targets.

To date, there is no recognized cure for Parkinson's disease. Aptamer-based immunotherapy is an attractive alternative. Researchers in China have reported preliminary results for a selective aptamer to α-synuclein. The purified human αsyn was used as the target for in vitro selection of aptamers using systematic evolution by exponential enrichment in Zheng et al.'s study [25]. This resulted in the identification of two 58-base DNA aptamers with a high binding affinity and good specificity to the α-syn. Both aptamers could effectively reduce α-syn aggregation in vitro and in cells and target the α-syn to intracellular degradation through the lysosomal pathway. In vitro, the aptamer inhibited the accumulation of αsynuclein and its association with the mitochondria [26]. It also induced intracellular α-synuclein degradation, and the neuron maintained viability despite overexpression of α-synuclein. In vivo, the a-synuclein aptamer can potentially inhibit the accumulation of a-synuclein in the cell while at the same time promoting the destruction of existing aggregates and reducing the toxic effects of α-synuclein aggregates on neurons. These effects consequently rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression [26].

Numerous examples in the literature have shown the efficacy of aptamers against several important targets. Aptamers have been developed to bind to αsynuclein monomers or its oligomer. These aptamers recognized β-sheet structure, the moiety though which they can bind not only to α-synuclein oligomer but also Aβ oligomer. This indicates that these aptamers could also potentially be deployed as drugs treat Parkinson's and Alzheimer's diseases (**Figure 3**).

activator, tPA, is a viable option in only 5% of stroke treatment cases, TLR4 blocking aptamers have been shown to be a promising and nontoxic alternative. Signal cascades play an important role in various aspects of cellular homeostasis and are also connected to several diseases [3, 31]. AMPA receptors are involved in excitatory synaptic transmission in the CNS and contribute to synaptic plasticity, as it is known in learning and memory processes [32]. They are hetero-oligomeric proteins, constituted of different combinations of four subunits GluR1-R4. The phosphorylation of GluR1 at the amino acid residues Ser831, Ser845, and Ser818 located at the receptor's intracellular domain has been found to have a strong impact on AMPAmediated neurotransmission. Liu et al. identified an RNA aptamer, termed A2, which modulates the phosphorylation of the serine residue Ser845 of the GluR1, whereas the phosphorylation of the Ser831 and Ser818 has been found to be unaffected [33]. Another work identified an RNA aptamer, named C5, which specifically binds and

*Aptamers targeting α-Syn oligomers for diagnosing and preventing onset of PD and dopamine for diagnosing*

**Figure 3.**

**279**

*dopamine concentrations [26].*

*Aptamers and Possible Effects on Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89621*

inhibits the mitogen-activated protein kinase (MAPK) Erk1/2 [34].

healthcare costs, and high quality of life in aptamers.

Aptamers may be of therapeutic use in treating neurological diseases. One of its biggest advantages is that aptamers are better able to penetrate tissues, cells, and blood-brain barrier (BBB) because they are small. They are essentially nonimmunogenic and chemically synthesized. Therefore, they eliminate concerns about biological contamination or long-term reagent formation that is often encountered in antibody treatments. Although new drug therapies are a risk to follow, there is a great potential in terms of increasing survival rates, decreasing

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disorder of the nervous system. Currently, there is no cure for MS, and the available medications only shorten the duration of attacks to slow the progression of the disease. Remyelination is a naturally occurring process in the body to restore damaged myelin sheaths after an MS attack. Rozenblum et al. identified a 40-nucleotide DNA aptamer which exhibits affinity towards murine myelin and binds to multiple myelin components in vitro [27]. In mice, it has been shown that aptamer is introduced into CNS tissue by intraperitoneal (IP) injection and dispersed in the tissue [28]. In addition, the aptamer allowed remyelination of CNS lesions in mice infected with Theiler virus [29]. Therefore, this aptamer can be used for recovery following an episode of MS and may alleviate the symptoms of MS.

The toll-like receptor 4 (TLR4) plays a crucial role in the adaptive immune response. It plays a role in many pathologies including stroke, myocardial infarction, atherosclerosis, sepsis, multiple sclerosis, and chronic pain. A research group from Spain investigated TLR4 blocking DNA aptamers, especially for the treatment of stroke. In an in vivo study involving mice and rats exposed to permanent middle cerebral artery occlusion (pMCAO), the TLR4-blocking aptamer reduced ischemic brain injury 4–6 h after injury [30]. The presence of aptamer in the blood and brain has been demonstrated by imaging studies [30]. Although tissue plasminogen

*Aptamers and Possible Effects on Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89621*

#### **Figure 3.**

aptamer was successful in neutralization in an in vitro study revealed the possibility

Parkinson's disease (PD), the second most common neurodegenerative disease after AD, affects over 7 million people worldwide. The pathology is characterized by loss of dopaminergic neurons, leading to decreased production of dopamine, a neurotransmitter that regulates movement and cognition. In the previous immuno-

established α-syn protein as an effective target for neuronal cell death. The pathogenesis of Parkinson's disease involves the accumulation of α-synuclein protein in neurons. Anti-α-synuclein antibody treatment has achieved some success. However, this antibody-based immunotherapy is limited by the inherent immunogenicity of antibodies and the inability of antibodies to reach intracellular targets. To date, there is no recognized cure for Parkinson's disease. Aptamer-based immunotherapy is an attractive alternative. Researchers in China have reported preliminary results for a selective aptamer to α-synuclein. The purified human αsyn was used as the target for in vitro selection of aptamers using systematic evolution by exponential enrichment in Zheng et al.'s study [25]. This resulted in the identification of two 58-base DNA aptamers with a high binding affinity and good specificity to the α-syn. Both aptamers could effectively reduce α-syn aggregation in vitro and in cells and target the α-syn to intracellular degradation through the lysosomal pathway. In vitro, the aptamer inhibited the accumulation of αsynuclein and its association with the mitochondria [26]. It also induced intracellu-

therapy, targeting the α-syn in PD models with monoclonal antibodies has

lar α-synuclein degradation, and the neuron maintained viability despite overexpression of α-synuclein. In vivo, the a-synuclein aptamer can potentially inhibit the accumulation of a-synuclein in the cell while at the same time promoting the destruction of existing aggregates and reducing the toxic effects of α-synuclein aggregates on neurons. These effects consequently rescued the mitochondrial dys-

Numerous examples in the literature have shown the efficacy of aptamers against several important targets. Aptamers have been developed to bind to αsynuclein monomers or its oligomer. These aptamers recognized β-sheet structure, the moiety though which they can bind not only to α-synuclein oligomer but also Aβ oligomer. This indicates that these aptamers could also potentially be deployed as

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disorder of the nervous system. Currently, there is no cure for MS, and the available medications only shorten the duration of attacks to slow the progression of the disease. Remyelination is a naturally occurring process in the body to restore damaged myelin sheaths after an MS attack. Rozenblum et al. identified a 40-nucleotide DNA aptamer which exhibits affinity towards murine myelin and binds to multiple myelin components in vitro [27]. In mice, it has been shown that aptamer is introduced into CNS tissue by intraperitoneal (IP) injection and dispersed in the tissue [28]. In addition, the aptamer allowed remyelination of CNS lesions in mice infected with Theiler virus [29]. Therefore, this aptamer can be used for recovery

function and cellular defects caused by α-syn overexpression [26].

drugs treat Parkinson's and Alzheimer's diseases (**Figure 3**).

following an episode of MS and may alleviate the symptoms of MS.

**278**

The toll-like receptor 4 (TLR4) plays a crucial role in the adaptive immune response. It plays a role in many pathologies including stroke, myocardial infarction, atherosclerosis, sepsis, multiple sclerosis, and chronic pain. A research group from Spain investigated TLR4 blocking DNA aptamers, especially for the treatment of stroke. In an in vivo study involving mice and rats exposed to permanent middle cerebral artery occlusion (pMCAO), the TLR4-blocking aptamer reduced ischemic brain injury 4–6 h after injury [30]. The presence of aptamer in the blood and brain has been demonstrated by imaging studies [30]. Although tissue plasminogen

of being a potential therapeutic tool for dementia patients.

*Neuroprotection - New Approaches and Prospects*

*Aptamers targeting α-Syn oligomers for diagnosing and preventing onset of PD and dopamine for diagnosing dopamine concentrations [26].*

activator, tPA, is a viable option in only 5% of stroke treatment cases, TLR4 blocking aptamers have been shown to be a promising and nontoxic alternative.

Signal cascades play an important role in various aspects of cellular homeostasis and are also connected to several diseases [3, 31]. AMPA receptors are involved in excitatory synaptic transmission in the CNS and contribute to synaptic plasticity, as it is known in learning and memory processes [32]. They are hetero-oligomeric proteins, constituted of different combinations of four subunits GluR1-R4. The phosphorylation of GluR1 at the amino acid residues Ser831, Ser845, and Ser818 located at the receptor's intracellular domain has been found to have a strong impact on AMPAmediated neurotransmission. Liu et al. identified an RNA aptamer, termed A2, which modulates the phosphorylation of the serine residue Ser845 of the GluR1, whereas the phosphorylation of the Ser831 and Ser818 has been found to be unaffected [33]. Another work identified an RNA aptamer, named C5, which specifically binds and inhibits the mitogen-activated protein kinase (MAPK) Erk1/2 [34].

Aptamers may be of therapeutic use in treating neurological diseases. One of its biggest advantages is that aptamers are better able to penetrate tissues, cells, and blood-brain barrier (BBB) because they are small. They are essentially nonimmunogenic and chemically synthesized. Therefore, they eliminate concerns about biological contamination or long-term reagent formation that is often encountered in antibody treatments. Although new drug therapies are a risk to follow, there is a great potential in terms of increasing survival rates, decreasing healthcare costs, and high quality of life in aptamers.
