Aptamer and its applications in neurodegenerative diseases

Abstract

Aptamers are small single-stranded DNA or RNA oligonucleotide fragments or small peptides, which can bind to targets by high affinity and specificity. Because aptamers are specific, non-immunogenic and non-toxic, they are ideal materials for clinical applications. Neurodegenerative disorders are ravaging the lives of patients. Even though the mechanism of these diseases is still elusive, they are mainly characterized by the accumulation of misfolded proteins in the central nervous system. So it is essential to develop potential measures to slow down or prevent the onset of these diseases. With the advancements of the technologies, aptamers have opened up new areas in this research field. Aptamers could bind with these related target proteins to interrupt their accumulation, subsequently blocking or preventing the process of neurodegenerative diseases. This review presents recent advances in the aptamer generation and its merits and limitations, with emphasis on its applications in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathy, Huntington’s disease and multiple sclerosis.

Introduction

Aptamers are single-stranded DNA or RNA nucleotides having a length of 40–100 mers. Recently, they have been extended to small peptides with 10–30 amino acid residues [1–4]. The name of aptamer originally comes from Latin “aptus” which means “fit” [5]. These single strands of nucleotides or peptides can specifically recognize and bind to a great deal of target molecules, such as metal ions, organic dyes and amino acids [6–8], antibodies [9], proteins [10, 11], whole cells [12–14], organs [15], viruses and bacteria [16, 17], by various intermolecular forces, such as van der Waals forces, hydrogen bonding, base stacking, etc. During the past 30 years, over 2000 aptamers have been developed to bind to about 141 target ligands [18]. Aptamers, considered as chemical antibodies, have more advantages over classical antibodies, since they have long half life, small size, low or no toxicity, and low immunogenicity, can be fastly and inexpensively produced [19]. Due to these merits, aptamers have attracted worldwide attentions for the diagnosis and therapy of various diseases.

Currently, aptamers have been applied in clinical diseases, such as cancer and inflammatory diseases, etc. [16, 20]. Pegaptanib, also called as Macugen, is the first aptamer approved by the US Food and Drug Administration to treat ocular neovascularization in December 2004. Meanwhile, it represents a milestone in drug development, because it is the first aptamer which is successfully used as a therapeutic agent in human beings [21–23]. Meanwhile, to be more interesting, aptamers have also been widely applied in food quality and safety monitoring [24].

Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), transmissible spongiform encephalopathy (TSE), Huntington’s disease (HD) and multiple sclerosis (MS), are plaguing people’s lives. Although the etiology of these neurodegenerative diseases is still elusive, there is a common pathological feature that is the accumulation of misfolded proteins. At present, which type of these misfolded protein, such as monomers, oligomers and fibrils, are toxic remains controversial. However, the predominant opinion is that oligomers are more toxic than others. Hence, preventing misfolded proteins accumulating as oligomers should be a good approach for treating the neurodegenerative diseases. Because aptamers just have the feature to bind with proteins to block their functions and accumulation, scientists are trying to use aptamers to treat disease or detect the pathologic processes. This paper mainly focuses on recent advances in the aptamer generation and its merits and limitations, with emphasis on its applications in neurodegenerative diseases.

Generation of aptamer

Aptamers were initially considered as DNA or RNA fragments. They were first developed almost simultaneously by two research groups of Tuerk and Ellington. In late 1990, Tuerk and Gold selected one eight-base RNA ligand which could interact with the bacteriophage T4 DNA polymerase [25]. Meanwhile, Ellington and Szostak isolated RNA molecules which could bind to a variety of organic dyes [5]. Recently, the term of aptamer has been extended to peptides which are selected by yeast-two-hybrid procedure or peptide array analysis [26–29]. In 1996, Pierre Colas et al. first generated peptide aptamers which could recognize and inhibit cuclin-dependent kinase 2 (Cdk2) [4].

Aptamers are generally screened out by systematic evolution of ligands by exponential enrichment (SELEX), which is a combinatorial chemistry technique of screening specific ligands by repeated rounds of partition and amplification from a large nucleic acid library containing 10¹⁴–10¹⁶ different candidates [25, 30]. Although SELEX has emerged as the most commonly used name for the procedure, some researchers also referred to it as SAAB (selected and amplified binding site) or CAST (cyclic amplification and selection of targets) [31, 32]. The traditional SELEX largely includes two processes, namely selecting aptamers with affinity to targets, and amplifying the bound aptamers (Fig. 1a). Technically, the processes begin with the synthesis of a nucleic acid library, in which the oligonucleotide is composed of the random sequences in the middle, flanked by fixed sequences [33]. Then, the library is incubated with the target molecules for a period of time under appropriate buffer and temperature conditions. The candidates with affinity can bind to the targets, while others with no affinity are still in free state. After washing the unbound nucleic acids from the binding molecules, the bound molecules are amplified by PCR. In order to achieve single-stranded DNA, there is a plethora of methods, such as asymmetric PCR, lambda exonuclease digestion, biotin-streptavidin separation, size separation on denaturing-urea PAGE and so on. For RNA aptamers, a transcription step by RNA polymerase is needed [34, 35]. Then these single-stranded molecules are used in the next selecting cycle. In general, it needs about 8–20 cycles in the entire process, until the sequences with high affinity and specificity are enriched [36]. Many aptamers were selected by the traditional SELEX in fact, such as amyloid β-protein (Aβ) and alpha-synuclein (α-Syn) (which were the major markers of AD and PD, respectively) [37, 38].

Fig. 1.

The processes of traditional SELEX, CE-SELEX, magnetic bead-based SELEX, and cell-SELEX. a The processes of traditional SELEX. It begins with the synthesis of a nucleic acid library. Then, the library is incubated with the target molecules for a period of time. The candidates with affinity can bind to the targets. Washing the unbound nucleic acids, the remaining binding molecules are amplified by PCR or RT-PCR. Then the molecules achieved from PCR or RT-PCR are used in the next cycle. b The processes of CE-SELEX. The nucleic acid library is incubated with the target molecules to form the mixtures. Then the mixtures are injected into the capillary. The bound nucleic acids are separated from unbound sequences through free solution capillary electrophoresis based on a mobility shift. c The processes of magnetic bead-based SELEX. The target molecules were immobilized on magnetic beads. Nucleic acid library is incubated with the target molecules on magnetic beads. Then the bound sequences are separated from unbound by magnetic force. d The processes of cell-SELEX. The nucleic acid library is incubated with negative cells which are not related to the target cells. Then, the unbound nucleic acids are collected and incubated with positive cells

Since the classical SELEX method for aptamer selection was developed, various improvements and modifications have been introduced to greatly shorten selection time and improve binding affinity [39, 40]. Among these modified SELEX methods, CE-SELEX, magnetic bead-based SELEX, automated SELEX and cell-SELEX are most frequently used. CE-SELEX (Fig. 1b), was first developed by Mendosa and Bowser in 2004 [41, 42]. It can increase separation power and reduce the nonspecific binding because of performing in free solution. So far, many aptamers have been generated by CE-SELEX, including aptamers against IgE [42], protein kinase C and so on [10]. Magnetic bead-based SELEX (Fig. 1c), was first developed by Bruno in 1997 [43]. It can enhance the binding affinity owing to being performed on the surface of the magnetic beads. Besides, the sample volumes only need 50–100 μl of sample [24, 44]. Some aptamers were also developed by this method, such as aptamers against 4-chloroaniline, mammalian prion protein (the major protein of transmissible spongiform encephalopathy) and dopamine (a key molecule in Parkinson’s disease) [39, 45]. Automated SELEX was reported by the group of J. Colin Cox in 1998 based on the Beckman Biomek 2000 pipetting robot [46, 47]. It can reduce the number of selection round to eight, and can complete about 12 select rounds in only 2 days, which means the selection efficiency is greatly improved. Ellington et al. [48] first adapted automated workstations to select anti-protein aptamers. Spiegelmers, RNA aptamers against the mirror image configuration of substance P, were also selected by this method. During this process, the aptamers were selected among a normal RNA pool against d-substance P and then the final selected aptameric sequence was synthesized by l-ribonucleic acid units. Consequently, they could bind to the naturally occurring l-substance P with comparable affinity [46].

Above three SELEX methods are generally used to screen aptamers for definite moleculars, latterly the method to select aptamers against cells comes to be developed. Cell-SELEX (Fig. 1d), first reported in 2003, is designed to target whole living cells, such as cancer cells [24, 49]. The process of cell-SELEX is easy, fast, straightforward, and reproducible. Numerous aptamers against various cancer cells were selected by this method. Aptamers, selected through cell-SELEX by the group of Weihong Tan, could specifically recognize leukemia cells [14]. In addition, there are other aptamers which can recognize the target cells derived from mouse tumor endothelial cells, glioblastoma multi-forme, prostate cancer, colorectal cancer etc. [50–53]. However, these cell-SELEX is based on the two-dimensional (2D) cell culture which has a limitation that cells could not grow in all directions as in vivo. Recently the three-dimensional (3D) cell culture was developed, it could mimic the microenvironment in vivo and cells are permitted to grow in all dimensions, so a novel method, 3D cell-SELEX, was introduced to select aptamers against cellular and extracellular targets. Eight aptamers, against PC-3 prostate cancer cell line, were selected through 3D cell-SELEX by the group of Aline G. Souza in 2016 [54, 55].

Merits of aptamer

Generally, the aptamers are considered as “chemical antibodies” because of their similar functions between aptamers and classical antibodies. Despite their similarity in functions, aptamers have more advantages than classical antibodies (Table 1 for detail), which drives aptamers to be widely used in clinic [19, 56]. First of all, the molecular weight of aptamers is about 5–10 kDa, which is lower than that of classical antibodies (approximately 150 kDa), so aptamers can reach the intracellular target molecules more easily than antibodies [30, 57]. Second, they can be easily generated by chemical synthesis, while the classical antibodies may be obtained with tedious work. Third, they are neither immunogenic nor toxic, while the antibodies are immunogenic, which makes aptamers to be ideal strategies in the treatment of immune diseases. Fourth, aptamers can restore the original structure easily within minutes once denatured, while the antibodies cannot, which indicates aptamers are stable for long time and could be transported at ambient temperature [30]. Fifth, the dissociation constant of most aptamers is as low as the picomolar–femtomolar (pM) level, which suggests aptamers might have more affinity to targets [58]. Sixth, aptamers can be easily labeled and adjusted, which indicates that aptamers can be easily modified according to the specified requirement [59]. Moreover, aptamers can distinguish closely related, but not identical proteins based on their different functions and structures, while classical antibodies cannot [60].

Table 1.

Comparison of properties of aptamer with antibody

Aptamer	Antibody
Smaller size allowing more efficiently entry into biological compartments	Large size limiting bioavailability or preventing access to many biological compartments
Produced chemically in a readily scalable process	Produced biologically in a difficult process
Non-immunogenic	Immunogenic
Able to select for specific targets	Limited ability to utilize negative selection pressure or to select against cell-surface
Production process is not prone to viral or bacterial contamination	Targets not available in functional recombinant form
Can usually be reversibly denatured	Susceptible to irreversible denaturation
Conjugation chemistries for the attachment of functional groups are orthogonal and can be readily introduced during synthesis	Chemistries required for the attachment of conjugation partners are stochastic and lead to product mixtures and reduced activity

Due to these properties, aptamers might be potential tools broadly applied in clinic. To date, they have been used for the study of many diseases in which cancer is the most widely treated, as shown in Table 2.

Table 2.

Examples of aptamers applied in cancer

Aptamer	Target	Cancer	References
AS1411	Nucleolin	Myeloid leukemia	[3]
A9, A10	PMSA	Prostate cancer	[6]
MUC 1 apt	MUC1	Breast cancer	[130]
TD50	Whole cells	Pamos cells	[131]
NOX-A12	CXCL12	Lymphoma multiple myeloma	[132]
TTA1	Tenascin-C	Glioblastoma	[133]

The limitations of aptamers and their optimization approaches

The limitations of aptamers

Despite the advantages mentioned above, aptamers still have obvious limitations which may hinder their use in clinical application [61–63]. The first serious limitation is the stability of unmodified aptamers, especially RNA aptamers, since these aptamers can be degraded by nucleases in blood [63]. The average half-life time ranges from several minutes to several tens of minutes. Such a short time range is not acceptable for most applications of aptamers in clinic. Then the fast clearance of aptamers also limits their therapeutic applications. The molecular weight of most aptamers ranges from 5 to 10 kDa, so they can rapidly penetrate into target tissue or tumor. However, they can also be rapidly cleared from the bloodstream before playing their roles in the clinical application [46, 64, 65].

The optimization approaches

To overcome the obstacles of aptamers in clinic, it is very necessary to develop approaches to enhance their stability as well as slow down their clearance. Recently, in order to ease the clinical application, various approaches have been developed, such as aptamer-chemical modification, aptamer–nanoparticle conjugation and so on.

Chemical modifications of aptamers

To improve the stability of aptamers, some chemical modifications have been used upon the aptamers such as 2′-fluoro-pyrimidine [66], 2′-O-methyl nucleotides [67, 68], locked nucleic acid (LNA) [67, 69], 2′-amino pyrimidines, 4′-thio pyrimidines [62, 65], or capping at the 3′- or 5′-termini with polyethylene glycol (PEG) [70], biotin-streptavidin [71], cholesterol [72] and so on. In 2003, 2′-fluoro-modified RNA aptamers, against prion protein which was an important protein in transmissible spongiform encephalopathy, were selected [66]. And in 2004, 2′-fluoro-pyrimidine was taken as a modification factor to develop Pegaptanib, which is the first aptamer approved by the US Food and Drug Administration to treat ocular age-related macular degeneration (AMD) [73, 74]. In 2006, two reports demonstrated aptamers with 2′-fluoro-pyrimidine against prostate-specific membrane antigen (PSMA) were used to target LNCaP cells expressing PSMA. The results indicated 2′-fluoro-pyrimidine can improve the aptamers stability [75, 76]. Moreover, LNA, a class of nucleic acid analogue with an extra bridge connecting the 2′-O and 4′-C in the ribose moiety, can improve the stability of aptameric sequences against nuclease digestion [69]. Kathrin S. Schmidt et al. demonstrated that LNA modification within aptamer TTA1 (recognizing human Tenascin-C) improved aptamers stability in 2004 [65].

Aptamer–nanoparticle conjugation

In addition to chemical modifications, nanoparticles conjugation to aptamers has also been tried to enhance the stability and affinity [77–79]. Nanoparticles are particles whose sizes are generally between 1 and 100 nm. They have unique virtues, such as the small size, the large ratio of surface area to mass, and the high reactivity [80, 81]. Due to these merits, nanoparticles have emerged as a novel tool to conjugate to aptamers to enhance aptamer stability and deliver aptamers to target tissue, such as liposomes, peptides, quantum dots, and virus-based vectors. Nanoparticle–aptamer (NP-Apt) bioconjugates can also improve the affinity of the aptamer. Farokhzad et al. identified that the conjugation of nanoparticle to A10 aptamer (which can bind PSMA transmembrane protein) could enhance the affinity between aptamer and the target. Moreover, they are the first to develop nanoparticle–aptamer bioconjugates to deliver targeted drugs in vitro [82, 83].

Besides, the aptamer–nanoparticle biosensor is another magical tool to recognize targets with high affinity. An aptamer–nanoparticle strip biosensor was developed by Liu et al. for the rapid, sensitive, and low-cost detection of cancer cells [84]. An AS1411 aptamer-functionalized nanoparticulate drug delivery system can prolong the circulation and accumulation of the drug in the target site of tumor, which can further inhibit tumor more efficiently and prolong mice survival [85]. A nanoelectronic biosensor for detecting dopamine (DA, a significant molecule in PD) modified by DNA–aptamer was developed by the group of Chen. This biosensor could improve and enhance the efficiency [86].

Other approaches

Except the two approaches mentioned above, there are also other means to overcome the limitations of aptamers. A great number of studies have proved that some vectors, such as polyethyleneimine (PEI), polycation, liposomes, chitosan, peptides, as well as quantum dots, could protect aptamer from degradation and improve its delivery efficiently [87–91]. Gong et al. first synthesized the complex of PEI/aptamer for cancer imaging in vivo. They were surprised to find PEI could successfully prevent TD05 aptamers from degradation at the N/P ratio from 3.8 to 15, without affecting the specific recognition of Ramos cells. Moreover, PEI/aptamer complex could more efficiently target passive tumor compared with free aptamer in mice bearing tumors. Hence, the PEI/aptamer complex may be a novel strategy to improve the stability of aptamers and to deliver aptamers to targets [87].

The applications of aptamer in neurodegenerative diseases

Neurodegenerative disorders have attracted worldwide attentions because of prevalent incidence in the upcoming aging society. These diseases, including AD, PD, TSE, HD and MS, are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Therefore, novel approaches to slow down or prevent these proteins accumulating would be potential approaches to treat these diseases. Aptamers could bind with these target proteins to prevent aggregating of the misfolded proteins or decrease the negative effects, which can further cure diseases or detect the pathologic processes.

Aptamers and AD

With the great increase of the human life span in the world, AD, one of the major diseases of the elderly progressive neurodegenerative diseases, is rapidly becoming a heavy burden and challenge in many countries [92]. The hallmark of AD is the misfolding of Aβ which is generated by sequential cleavage of amyloid precursor protein (APP). Aβ aggregation may disrupt the normal physiological functions of cells, inducing oxidative stress and nerve inflammation, which could further lead to the dysfunctions of cells, even apoptosis and death (Fig. 2). Therefore, Aβ has been a target for the diagnostic and therapeutic reagents of AD.

Fig. 2.

The damage of the aggregations of misfold proteins. Aβ and α-Syn are a class of proteins which are prone to aggregate and fold. The aggregations can disrupt the normal functions of cells and proteins, such as the impairment of the degradation systems, the dysfunction of mitochondria, the generation of oxidative stress, which could further lead to the disfunctions of cells, even apoptosis and death

Since Aβ aggregation is toxic, firstly it is a good idea to reduce Aβ production. As is known to all, the generation of Aβ is triggered by β-site APP cleaving enzyme-1 (BACE1). Thus, BACE1 should be a worthy target of the interference of Aβ production and treatment of AD (Fig. 3) [93]. One DNA aptamer, called A1 selected by the group of Liang et al. may bind to BACE1 with high affinity and good specificity. Consequently, A1 had the potential to decrease the production of Aβ40 and Aβ42 [94]. Furthermore, RNA aptamers against a short cytoplasmic tail (B1-CT) of BACE1 also have been selected by Rentmeister et al. [95]. These RNA aptamers can specifically bind to B1-CT without affecting other important biological activities. So they could be potentially applied to prevent or slowdown the onset of AD.

Fig. 3.

The aptamers (apt) against the AD targets. The hallmark of AD is Aβ of which the oligomer will disrupt the normal functions of cells. BACE1 can trigger the generation of Aβ, and B1-CT is a tail of BACE1. PrP^C is a receptor of Aβ oligomer. The aptamers against Aβ oligomers, B1-CT and PrP^C might be used as tools to diagnosis the Aβ oligomers and even prevent the generation of Aβ, which can decrease the serious effects to the cells

As Aβ oligomer is toxic, the aptamer directly binding to oligomer may also prevent the toxicity (Fig. 3). RNA aptamers against Aβ40 oligomer were selected by Bitan et al. in 2009. However, the data showed that aptamers had low specificity to Aβ oligomer [96]. A DNA aptamer, which can specifically recognize and bind to Aβ oligomer, was generated in 2012. Since these aptamers are specific for oligomers, they could be more efficient and specific tools than antibodies in the application of recognizing the Aβ oligomer, preventing Aβ toxicity, as well as diagnosing AD [37, 38].

Since Aβ oligomer is toxic, there should be definite receptors to exert its toxic role. In this scenario, it may be a good choice to block the target receptor of Aβ oligomer (Fig. 3). It is found that the normal prion protein (PrP^C) is one of the target receptors of Aβ oligomer, and Aβ-oligomer could bind to PrP^C with affinity [97, 98]. So PrP^C may be a mediator of Aβ-oligomer to induce synaptic dysfunction, and aptamers against PrP^C can prevent Aβ-oligomer binding to PrP^C and rescue synaptic plasticity, even treat AD [97].

Aptamers and PD

PD is the second most common neurodegenerative disease mainly affecting 1 % of individuals above the age of 60. It is primarily defined by symptoms of motor impairment, such as resting tremor, bradykinesia, postural instability, rigidity, and so on [99–102]. The pathology of PD is characterized by the presence of inclusions termed Lewy bodies (LBs) in neurons, and the selective loss of dopaminergic neurons in the substantia nigra pars compacta [103]. The major protein component of LBs is the presynaptic protein α-Syn which is misfolded and aggregated [104]. The aggregations of α-Syn, especially the oligomers, have negative roles in the neurons, such as the impairment of the degradation systems, the damage of mitochondria, the generation of oxidative stress and toxicity (Fig. 2) [105–108]. Hence, aptamers against α-Syn would be potential tools to diagnose or treat PD.

Predominantly, α-Syn oligomers are more toxic than monomers and fibrils, so oligomers are seen as the key targets of PD. The first aptamer against α-Syn oligomers, called “M5-15”, was selected by the group of Ikebukuro in 2010. But surprisingly, M5-15 could also slightly bind to α-Syn monomers, which indicates that M5-15 has low specificity to α-Syn oligomers [109]. Two years later, they developed another eight DNA aptamers against α-Syn oligomers. Interestingly, these aptamers definitely recognize the β-sheet structure [38], namely they can bind to not only α-Syn oligomer, but also Aβ oligomer, which indicated these aptamers could also be employed as magical drugs to treat even cure PD and AD (Fig. 4).

Fig. 4.

The aptamers (apt) against the targets of PD. The pathology of PD is characterized by the presence of inclusions termed LBs whose major protein component is α-Syn, and the selective loss of dopaminergic neurons in the SN. α-Syn oligomers can also disrupt the normal functions of cells. So aptamers targeting α-Syn oligomers could be developed as useful tools for diagnosing PD or preventing the onset of PD. DA is another target, and aptamers against DA can diagnose the concentration of DA

Interestingly, the peptide (GVLYVGSKTR), derived from β-Syn protein, could bind to α-Syn and inhibit its aggregating found by the group of Shaltiel-Karyo. This peptide could reduce α-Syn amyloid fibril and soluble oligomer in vitro. Strikingly, administering this peptide to a Drosophila PD model, expressing A53T α-Syn, could ameliorate the behavioral defects and reduce α-Syn accumulation in the brains [26]. Hence, this peptide should be another potential way to prevent the onset of PD.

Not only the presence of LBs, PD is also characterized by the selective loss of DA in the substantia nigra. DA is a small molecule with weight of 189 Da, and it is involved in neuronal signal transduction. The loss of DA would lead to the deregulation of basal ganglia which further causes motor symptoms and non-motor symptoms [110]. As we all know, the concentration of DA usually ranges from 10⁻⁸ to 10⁻⁶ M in the normal physiological environment [111]. The deficiency of DA neurotransmission may be implicated in PD [112, 113]. Accordingly, the aptamers against DA may be another tool to monitor DA concentration and study the pathological mechanism of PD. The first RNA aptamer against DA called dopa2, selected by Tocchini-Valentini et al. [45], could bind to DA with high affinity and specificity. Then a novel label-free electrochemical aptasensor for detecting DA was developed by Liu et al. in 2012. The aptasensor has successfully tested the DA of human serum [114]. Another RNA aptamer-based electrochemical biosensor for selective and label-free analysis of DA was reported by Farjami et al. [115]. To further enhance the detecting accuracy, Chen et al. developed a new nanoelectronic device as a biosensor for detecting DA by modifying the DNA–aptamers on a multiple-parallel-connected (MPC) silicon nanowire field-effect transistor. This biosensor can improve the detection limit, distinguish DA from analogues, as well as monitor the release of DA [86].

Aptamers and TSE

TSE is another class of neurodegenerative disorders, affecting human beings and other mammals [116]. The pathological characteristic of TSE is the conversion of normal cellular prion protein (PrP^C), an α-helix-rich isoform, to abnormal PrP^SC subtypes, a β-sheet-rich isoform, and accumulating in the brain [117]. Thus, diagnosing PrP^C and PrP^SC, as well as inhibiting the transformation from PrP^C into PrP^SC might be useful approaches to study the pathogenic of TSE and treat TSE. Recently, a great deal of aptamers have been selected against both PrP^C and PrP^SC and these aptamers are isoform- and species-specific.

SAF-93, 2′-fluoro-modified RNA oligomers, for PrP^Sc were selected in 2003 [66]. The affinity of these aptamers for PrP^Sc was more than tenfold higher than for PrP^C because of the existence of two specific heparin-binding sites within the PrP^Sc molecule. Moreover, SAF-93 could inhibit the conversion of PrP^C into PrP^Sc [66]. But because 2′-fluoro-modified SAF-93 are too long and complex, they are not ideal tools for the clinical diagnosis and therapy of TSE. Subsequently in 2004, James et al. identified the functional structures within SAF-93, and synthesized an aptamer SAF-93_{(1-34,35bioU,36-60)} to detect the PrP^Sc [118]. These aptamers targeting PrP^Sc could be potentially used in diagnosing and treating TSE.

Because TSE is often occurring in various animals, the aptamers against PrP should also be species-specific (Table 3). Aptamers against the mouse prion protein (mPrP) were isolated by the team of Nishikawa [119, 120]. One DNA aptamer which could bind to both recombinant and mammalian PrP^C but not PrP^Sc was selected by Takemura et al. [121]. In the same year, RM312, an RNA aptamer that could bind to sheep recombinant PrP, was selected by Mercey et al. [122]. Additionally, an RNA aptamer targeting bovine prion protein (bPrP) was selected by Mashima et al. [98]. So, species-specific aptamers might become novel tools to diagnose and treat TSE in various animals.

Table 3.

The species-specific aptamers against PrP

Name	Source	Target	References
60-3	RNA	Mouse PrP	[118]
	DNA	Mouse PrP	[119]
	DNA	Recombinant and mammalian PrPC	[121]
RM312	RNA	Sheep PrP	[122]
R12	RNA	Bovine PrP	[119]

Aptamers and HD

HD is a dominantly inherited disease in neuronal tissue. It is suggested this disorder has affected about 5–10 individuals per 100,000 [123]. Affected individuals always suffer from progressive motor as well as cognitive declines, such as the loss of awareness, depression, dementia and anxiety. The pathogenesis of HD seems to be the misfolding and aggregation of mHTT which is a form of mutant huntingtin protein [123, 124]. Hence, we must make efforts to find agents which could destroy or prevent the pathway of mHtt aggregation.

In 2006, Skogen et al. reported that an aptamer, 20-m G-rich oligonucleotides, could effectively inhibit HTT aggregating. This aptamer could enhance the viability of PC12 cells which overexpressed the mHTT fragment gene, which suggested it would likely to be new agents to study the mechanism of HD and treat this disease [125].

Aptamers and MS

MS is an autoimmune neurodegenerative disease with a prevalence of around 0.1 % in the Western world. The clinical features include visual loss, gait and cognitive impairment, fatigue, and so on. And pathological characteristics are the destruction of myelin required for electrical insulation of neuronal axons in the CNS [126, 127]. So the myelin may be a key material for the research for MS. Several aptamers against myelin have been selected by the SELEX.

A 40-nucleotide DNA aptamer against murine myelin was selected by Nastasijevic et al. in 2012. This aptamer could promote remyelination of CNS lesions in the MS mice model [128]. Then LJM-3064, a guanosine-rich 40-m DNA aptamer, was found to mediate remyelination in the mouse model of MS in 2013. And this aptamer could undergo an ion-dependent conformational switch, which provided important thermodynamic insights that may be an optimizing tool for developing the therapeutic remyelinating agent [127]. Another two aptamers for myelin were obtained by Rozenblum et al. through SELEX method in 2014 [129]. Moreover, one selecting aptamer was more sensitive than the commercial antibody. Therefore, these aptamers might be useful tools for medical research and treatment of MS.

Conclusion and perspective

Aptamers have gained great attention since their introduction in the early 1990s. Aptamers are a class of substances having similar functions to classical antibodies, but they have unambiguous advantages over classical antibodies, including small size, low or no toxicity, and low immunogenicity. Despite the advantages over classical antibodies, there are several barriers which limit the use of aptamers in clinical application, such as rapid degradation, the susceptibility to nucleases, easy renal filtration and so on. Fortunately, various approaches have been proposed to overcome these limitations such as aptamer-chemical modification, aptamer–nanoparticle conjugation and others.

The obvious merits have driven aptamers usage in clinic, such as cancer, eye and inflammatory diseases. Pegaptanib is the first and sole aptamer that has been successfully applied in the clinic, which is a milestone of aptamers to be used in the diseases. Many other aptamers are being developed which indicates more and more aptamers will be applied in the neurodegenerative disorders.

Neurodegenerative disorders are characterized by the accumulation of misfolded proteins in the CNS, including AD, PD, TSE, HD and MS. Aptamers against these targets which are associated with these diseases have been developed, and they are being applied in clinical trials. Along with the advancements such as nanotechnology, microfluidics, microarray, and others in the field of clinical diseases, aptamers will gain the special area in clinic application. Moreover, the successful developments in aptamers over the last 30 years are notable, which reveals aptamers are likely to become valuable drugs to cure these neurodegenerative diseases.