Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins

Abstract

Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another intersting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.

Introduction

Since 1990, a new group of nucleic acid based ligands were introduced that were able to interact with specific targets through the high affinity and selectivity [1, 2]. A wide range of various targets has been considered for aptamer development, from small molecules and ions to the large macromolecules and also whole cells or simple organisms [3]. Scientists were interested in protein specific aptamers as one of the most promising and applicable agents in science and medicine.

Numerous types of technologies have been developed for aptamers in the field of therapeutic and diagnostic applications, as well as the combination approach of therapeutic and diagnostic, termed “theranostic” [4, 5]. Another important focus in aptamer studies is their analytical applications, especially, in the design of biosensors [6–8], lab-on-chip detection systems [9–12] and protein purification [13].

Protein Specific Ligands

Peptides and proteins are a major group of biomolecules and play important roles in physiological and pathological processes in the living organisms, especially in Homo sapiens. Introducing selective technologies like monoclonal antibodies and aptamers in the field of protein research would help scientists to reveal new insights in science and technology.

Design and development of high affinity ligands for protein or peptide targets, include small molecules, peptide ligands, monoclonal antibodies and aptamers, expressed valuable potentials as therapeutic and analytical agents. Among the ligands, aptamers exhibited several advantages such as more selectivity and affinity compared to the small molecules, rather than the smaller size, more stability and diversity compared to the peptide ligands and monoclonal antibodies, and generally, simple and economically cost-effective selection process of aptamers [14]. Additionally, attachment of chemical or biological functional groups to the aptamers could be implemented simply, parallel to control the changes in aptamers’ structures and affinity [14–16].

At the other site, the detection of disease-related proteins in complex biological media demands sensitive, reliable, and also low-cost sensing methods. According to the stated advantages, such as small size, easy and uniform production, easy modification, good compatibility to the modern nanotechnology and easily labeling with variant signaling agents, aptamer based techniques can apply successfully in this regard [17]. One of the challenges in aptasensor development is obtaining proper signal for detection and quantification [18], that will be handled by different signal amplification techniques or accompanying nanoparticles potentials. Various strategies that have been studied in this regard, were enzyme-assisted fluorescence signal amplification by nicking enzyme and using a complementary oligonucleotide [19–21], synergetic amplification by a three-dimensional nano-scale catalase enzyme in combination to the DNA-platinum nanoparticles [22], applying sandwich approach (in different types of sensing methods like what applied in ELISA) [23, 24] and some others like DNAzyme [25], gold and silver nanoparticles, or quantum dots assembly [26, 27]. Through method development, a model protein is useful, which could finally be expanded to the other related comparable targets. Two of the most studied model proteins are thrombin [28–33] and lysozyme [34–38] which are considered as classical biological proteins.

Protein Specific Aptamer Selection Methods

Selection of protein specific aptamers have been done according to the classical method of “systematic evolution of ligands by exponential enrichment” (SELEX) (Fig. 1) and its derivatives or Non-SELEX methods that are relatively easy and non-expensive [14]. Major difference between SELEX and Non-SELEX methods is in the presence of the amplification step; by using Non-SELEX process, high resolution techniques such as capillary electrophoresis applied for the partitioning step. Consequently, by elimination of the amplification step, the duration of aptamer selection decrease, as well as aptamers with higher affinity are developed [39]. The important challenge in protein research is the conservation of preferred protein conformation during selection process and beside this, achievement to the anticipated biological function on the target. In the case of analytical aptamer application, the best choice is “no effect” on protein function and just selective binding of the oligonucleotide based affinity ligand is favorable. Generally, in the case of aptamer selection against proteins, considering proteins’ sensitivity to the instability conditions, using Non-SELEX methods might have beneficial features according to the fewer number and shorter run of SELEX process [40]. Nevertheless, various subtypes of SELEX have been developed and applied to protein specific aptamer selection (Fig. 2) [41–53]. Additionally, introduction of next generation sequencing techniques help the scientists to develop automated or semi-automated SELEX process, which resulted in simple and fast detection of high affinity selective aptameric ligands [54, 55] and their application in proteomics [56].

Fig. 1

Schematic presentation of classical SELEX process: in a classical SELEX process, five fundamental steps should be considered; at first, starting single stranded random oligonucleotide library is incubated with the target molecules (proteins) in a suitable incubation condition (pH, Temp. and buffering components). At step 2, bound oligonucleotides are separated from unbound oligonucleotides by a high resolution separation technique which partition the mixture according to the physiochemical features. At step 3, the bound oligonucleotides are dissociated from the oligo-target complex. Then, selected oligonucleotides are amplified, routinely by PCR. At final step, double stranded oligonucleotides from PCR process convert to the single stranded oligos and enter the next round of selection. In a complete SELEX process, one to three rounds of counter selection are performed to remove cross-binding to undesirable target molecules. In the counter selection, unbound oligos enter the next round of selection

Fig. 2

Aptamer selection against protein targets have been performed by various subtypes of SELEX [41–53]. Changes in oligonucleotide chemistry and the partitioning step of SELEX process could improve the selection feasibility or the selectivity and affinity of the final aptameric ligands. These changes result in different subtypes of SELEX process

In-vitro selection of aptamers could be done against diverse motifs in the protein structure. Similar to the antibodies’ binding site on protein structure named epitope, the binding site of aptamers called apatope [57]. There are various apatopes in the protein structure that could be targeted during a protein specific aptamer selection and accordingly, among in vitro selection studies, different aptamers would be developed for a single protein target, as it was indicated for thrombin with several aptameric ligands. In some studies, thrombin specific aptamer with dual binding capacity was also selected [54].

In order to aptamer development against cell surface protein by in vitro selection process, some types of modified SELEX process have been developed [58]. Cell-SELEX as a major subtype of SELEX was firstly developed in early twenty-first century against YPEN-1 endothelial cells [59] and U251 glioblastoma cells [60] to disclose surface antigens specific aptamers. Recently, some types of Cell-SELEX have been applied to make cell surface antigen specific aptamers include simple Cell-SELEX [61], Target expressed on cell surface (TECS) SELEX [62, 63], monoclonal surface display SELEX (MSD-SELEX) [64], Cell internalization SELEX for in vitro selection of molecules that internalize into cells [65]. The details of the different selection methods and related advantages and disadvantages were not the scope of this review; accordingly, they were just stated in a few words (Table 1).

Table 1 Advantages and disadvantages of different aptamer selection methods

Another important issue in aptamer development against protein targets is the consideration of post-translational modifications (PTM) of proteins. PTMs change the surface properties and as aptamers bind to the available surface of target, PTM and its detection by aptamers have important value in clinical and analytical applications. One of the PTMs is glycosylation that glycan units attach to the superfacial aminoacids of proteins. A specific method for in vitro aptamer selection was developed to prepare glycan-targeted aptamers, which named boronic acid-based aptamers (boronolectins) and allows focusing on the glycan substructure of glycoproteins. This method applied to fibrinogen as a model [66], prostate specific antigen (PSA) [67] and recombinant human erythropoietin (rHuEPO) [68], and could also be applicable for the development of aptamers against other glycoproducts, such as glycolipids and glycopeptides. Similarly, aptamers discriminate different PTMs of a protein can be valuable in diagnosis and disease therapy. Changes in the PTMs of a protein commonly results in diseases and thus, detection of the changes have great value in biomarker discovery. It was approved by a study on Cyclophilin B that have different PTM in healthy and pancreatic cancer patients and the protein specific aptamer could discriminate between these two groups [69].

Different Application of Proteins Specific Aptamers

Diagnosis Applications of Aptamers

Interaction of protein specific aptamers with their targets could increase the protein stability and resistance to denaturation, which make aptamers as new class of bio-recognition elements for therapeutic, diagnostic and sensing applications [81]. Biomarkers are defined by National Institute of Health (NIH) as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” and according to the World Health Organization (WHO), defined as “any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease” [82]. Discovery and validation of biomarkers is one of the most interesting fields of biological studies. There are diverse methods of proteomics that utilized for this purpose. Among them instrumental analytical technologies such as gel electrophoresis or mass spectroscopy are more established; however, they have limitations such as high cost, lack of enough specificity and selectivity. According to this challenges, antibody base methods like enzyme linked immune sorbent assay (ELISA) is applied for more sensitive analysis [83]. This type of assay also, showed some limitation in simultaneous detection of multiple targets; consequently, scientists introduced aptamer technology to address these problems and concurrent detection of multiple biomarkers hold great promise in disease diagnosis especially in cancers or inflammatory disease [37, 83, 84]; such as an electrochemical aptasensor for simultaneous detection of two important inflammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) [18].

Aptamers can be applicable in biomarker discovery or specific antigen detection from biological samples. Aptamers would be even selected against unknown targets, therefore it is possible to develop sample specific aptamers (solid or fluid biological samples), even in situation that the molecular components are not recognized [85, 86]. Using aptamer technology as a part of proteomics’ study results in biomarker recognition from a small amount of initial clinical samples, screening a large number of patients [79]. Additionally, as there are multiple diversities in pathology and disease pattern of different population, it would be possible to discover and customize population specific diagnostic or therapeutic agents. Screening of peptide or protein ligands for unknown targets becomes possible with aptamer technology [87].

Detection of early markers have important diagnostic value and precise biomarker detection during golden time could result in life saving. Aptamers can be valuable in this regard, such as the aptamer developed for myoglobin in cardiovascular disease [88], ApoA1 for early monitoring of hepatocellular carcinoma [89], CTAP III/NAP2 as indicator of lung cancer [90], retinol-binding protein-4 (RBP4), visceral adipose tissue-derived serpin (Vaspin), and nicotinamide phosphoribosyl transferase (Nampt/visfatin) adipokines for early diagnosis of type 2 diabetes [91, 92], vascular endothelial growth factor-165 (VEGF 165) as indicator of cancer or metastasis [93], mucin-1 (Muc-1) in epithelial cancers (breast, bladder, etc.) [94] alpha-methylacyl-CoA racemase (AMACR) as an emerging prostate cancer biomarker [47] and C reactive protein (CRP) for early diagnosis of sepsis [95]. Some other proteins are prognostic markers, like protein Ras for restenosis occurring after coronary surgery or angioplasty that its specific aptamer could be used for preventive or therapeutic goal [96].

Some of the aptasensors have been developed based on lysozyme as a protein model are presented in Table 2.

Table 2 Several applications of lysozyme specific aptamer

Cancer Cell Surface Biomarkers

One of the most important surface biomarkers are the cancer cells biomarkers. Numerous of these biomarkers have been previously identified by different proteomics studies; therefore, they could be a potential target for aptamer development. Biomarker specific aptamers could also be used for isolation or detection of cells from heterogeneous biological samples which is important in both basic biological research and clinical diagnostics [106]. According to the cell SELEX technology, it had become possible to develop aptamers specific to the cell surface markers like cancerous biomarkers. As a result, numerous aptasensors have been designed to diagnose different cancers. These aptamers can be developed against the known biomarker and to unknown biomarkers for its clinical application or investigational discoveries. Among the biomarker studies by aptamers, two different lanes have been considered. In one point of view, single biomarker was the target of aptamer selection or application design, but at the other side, a group of biomarkers discovered or were applied to achieve the clinical or analytical goal; such as what was done by Wu and coworkers to develop nano-sensors for simultaneous detection of tree types of cancerous cell lines [107]. In this regard, selective aptamers against gastric carcinoma cells were also developed by cell SELEX and showed good efficacy in cancer cell detection and imaging [108, 109].

One of the well-known cancer biomarker is cell adhesion receptors like epithelial cell adhesion molecules (EpCAM) and selectins. EpCAM is overexpressed in various solid tumors such as prostate and breast cancer and is an valuable antigen for clinical application in cancer diagnosis, prognosis, imaging, and therapy [110]. First EpCAM specific aptamer was presented by Shigdar et al. [111] to develop novel targeted cancer nanomedicine and molecular imaging agents. Up to date, some other studies have been focused on the specific aptamer development for this transmembrane glycoprotein [112, 113] and its application in chemotherapeutics drug delivery [114–116]. Another famous biomarker is mucin-1 that its specific aptamers are interesting tools for cancer diagnosis [117, 118] and drug delivery [119, 120]. The human mucin family with more than 20 proteins is divided to secreted mucins and membrane-tethered mucins [121]. Mucin-1 is a transmembrane glycoprotein that is expressed on the surface of epithelial cells and overexpressed or abnormally glycosylated during different tumor development [122, 123]. Its role in diagnosis of and drug delivery to epithelial cancers like ovarian cancer [124], colon cancer [125] and breast cancer [126–128] have been studied by aptamer technology.

Another cell surface receptor that overexpressed in malignancies is epidermal growth factor receptor (EGFR/HER1/c-ErbB1), observed in many solid cancers pathophysiology. Specific aptamer against EGFR was developed [129] and showed good potentials for cancer diagnosis [130], cancer therapy [131, 132] and targeted drug delivery [133].

Through some studies, a unknown mixture of targets was evaluated by aptamer technology and a specific single biomarker was discovered for more investigations; such as the discovery of stress-induced phosphoprotein 1 (STIP1). It was found that this protein could be a potential biomarker of ovarian cancer. It was also showed that its specific aptamer inhibited cell invasion [134].

Some other biomarkers are secreted to the circulating system or biological fluid and could be detected in the serum or other biological samples of patients rather than tissue sample. In this category, numerous aptasensors were developed such as sensors for prostate specific antigen (PSA), a circulating biomarker indicative of prostate cancer [135, 136], Platelet-derived growth factor-BB (PDGF-BB) that is often over expressed in human solid tumors as a marker of tumor angiogenesis [137, 138].

Vascular endothelial growth factor (VEGF) is also one of the serum markers to promote angiogenesis and related to the different diseases such as cancers, rheumatoid arthritis, psoriasis, and proliferating retinopathy. First FDA approved aptamer based therapeutic was an anti-VEGF aptamer used for the age related macular degenerative disease (AMD). Since the first report of anti-VEGF aptamer, there are several investigations about its various applications especially as sensors [93, 139–143].

Diabetes Markers as a Metabolic Disease

Diabetes is a health threatening disease that affects the health during long time and its treatment and early diagnostic have special importance in clinic. Insulin as the mostly important peptide in glucose uptake to the tissues/cells is an attractive target for aptamer development [143–145]. Designing aptasensors for detection of insulin is another interesting aspect of aptamer applications [146–148]. Insulin receptor specific aptamers could also have promising potentials in therapeutic strategies [149].

Another important peptide in glucose hemostasis is glucagon, which increases the plasma glucose level. Glucagon specific mixed DNA/RNA aptamers was developed to improve glucose tolerance and reduce insulin need in diabetic patients [150]. In addition, the role of Insulin like growth factor-1 aptamer in glucose hemostasis [151] and also, muscle injury and dystrophy [152] were studied.

Adipokines, as the other diabetes biomarkers that their level increases during glucose intolerance, were studied in early diabetes diagnosis [91, 92]. In the same way, detection of glycated hemoglobins (HbA1c) in comparison to the total hemoglobin (Hb) is an indicator of average blood glucose levels over the past 2 or 3 months. Therefore, it is a monitoring marker of the diabetes progress or treatment efficacy. Accordingly, an aptamer based sensitive microfluidic system was developed as a promising tool for cost-effective point-of—care diagnosis and also prognosis system [153].

Neurodegenerative Disease Specific Proteins

Neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and prion diseases involves metalloprotein misfolding and aggregation (amyloid beta, alpha synuclein, and prion protein) [154]. Aptamers as oligonucleotide affinity ligands serve promising tool in the neurodegenerative disease diagnosis or treatment [155, 156], for instance the amyloid beta specific aptamers in Alzheimer [157]. The aptamers could be used in targeted drug delivery systems [158] as used in delivery of curcumin to the Alzheimer plaques [159]. Additionally, using aptamer technology in the proteomics study and biomarker discovery for Alzheimer disease could assist researchers to develop valuable diagnosis method using more accessible body fluid samples like plasma [160].

The other factor that involves in a number of fatal neurodegenerative diseases in mammals is Prions. Prion is a protein in misfold state that as an infectious agent results in diseases such as mammalian transmissible spongiform encephalopathies, including bovine spongiform encephalopathy and scrapie in sheep. In humans, prions are also the cause of fatal infections [161]. Early diagnosis and preventive strategies are the most important therapeutic approach to face these invasive agents [162, 163]. Selection of its specific aptamer [163–166] and designing of various biosensors based on aptamer technology [167–170] have been presented the promising and applicable efforts to fight prion infection.

The other related protein target as diagnostic marker for aptamer selection is α-Synuclein, an unfolded protein that causes several neurodegenerative diseases such as dementia and Parkinson’s disease [171, 172]. Uses of aptamers and related technologies in neuroscience have been improved extensively.

Inflammatory Markers

Inflammatory system is activated during different disease process; accordingly, detection and modulation of inflammatory proteins play an important role in clinic. Aptamer-based techniques have been proved for the detection and measurement of inflammatory markers, such as a micro-cantilever sensor that developed for Lipocalin-2 (a biomarker for many inflammatory-based diseases, including acute kidney injury, cardiovascular stress, diabetes, and various cancers) [173].

Beside the cancer detection, Platelet-derived growth factor-BB (PDGF-BB) is also, one of the important cytokines involved in neural inflammation and its specific aptamers directed to the diagnosis of inflammatory episode like designing of aptasensors [174]. Generally, application of the aptamer and aptasensors in the field of inflammatory diseases is an attracting area for researchers and clinicians.

Cardiovascular System Specific Markers

Proteins have critical rule in function or malfunction of cardiovascular system. For example, coagulation factors are plasma proteins that involved in protection mechanism of the body against bleeding events. They are one of the most interesting target groups in aptamer development. Thrombin is the first protein that its selective aptamer was developed in 1992 [28]. Many of the novel aptamer related sensing technologies have been developed based on thrombin specific aptamer because they were studied and characterized exclusively (Table 3) [175].

Table 3 Some examples of aptasensors developed by thrombin specific aptamers

The analytical application for aptamers specific to cardiovascular proteins includes their usage in early diagnosis and prognosis of stroke events; such as aptamers against Ras protein [96] and myoglobin [48], as mentioned before.

Aptamers for Imaging

Aptamers due to the small size and the wide range of targets, are ideal targeting molecules for in vivo imaging [181]. Aptamers against cell surface proteins can be used in this regard, by delivering contrast material to target site [182]. The aptamer-based probes have been used for specific detection of biomarkers and cellular proteins on the surface of target cells [183].

The first report of protein aptamer for imaging was a radiolabeled aptamer against neutrophil elastase. This radiolabeled aptamer interacted with the elastase on the surface of neutrophils at the sites of inflammation [184, 185]. Aptamers can also be used for tumor imaging [186]. For example, aptamer bound to MUC1 protein, a large cell surface mucin glycoprotein highly expressed at the surface of some cancer cells, was radiolabeled with 99mTc for tumor imaging [187]. Aptamer selected against uMUC-1 (Under-glycosylated mucin-1), labeled by 68 Ga, has been developed for imaging tumor cells that widely express this antigen [188]. Radiolabeling of the HER2 specific aptamer with 99mTc for imaging of ovarian cancer cells [189] and Nucleolin aptamer for detection of lung cancer cells [190] are the other examples. Similarly, a radiolabeled aptamer for tumor imaging has been used against tenascin C, an extracellular matrix protein. This aptamer was labeled with 99mTc and used for imaging solid tumors that over-express this membrane protein including breast, glioblastoma, lung, and colon [191].

Some studies evaluated nanoparticles based anti-cancer drug delivery systems to diagnose and treat cancer, simultaneously. In this regard, daunorubicin–MUC1 aptamer–quantum dot—(DNR–MUC1–QDs) conjugates have been developed for cancer treatment and imaging of MUC1-positive prostate cancer cells as a theranostic agent [192]. Conjugated aptamer–gold nanoparticles have also been used for the detection of PSMA-expressing cancer cells by CT imaging [182]. EpCAM is another surface marker on human hepatic stem cells. EpCAM aptamer conjugates with an imaging agent combined to the potentials of nanoparticles have been used for imaging of cancer [193, 194]. Generally, the targeted delivery of contrast materials to target sites using aptamers is an effective approach in bioimaging and early detection of diseases.

Aptamers in Drug Delivery

A wide range of therapeutic molecules can be conjugate with cell surface biomarkers specific aptamers directly or by using nanoparticles in different drug delivery systems and delivered selectively to target cells [195, 196]. The use of aptamer bio-conjugate for targeted drug delivery increases drug internalization to the target cells and consequently, improves therapeutic efficacy [197]. In addition, aptamer-drug complex has lower side effects and cytotoxicity compared to drug alone, because of specific cellular uptake.

For example, conjugation of aptamer selected aganist CD117 biomarker (overexpressed on AML cells) with methotrexate, lead to targeted drug delivery and cell growth inhibition with minimal side effects [198]. In another study, for delivering daunorubicin to acute lymphoblastic leukemia cells (ALL), aptamer specific for protein tyrosine kinase-7, one cell surface over-expressed marker in leukemia, was conjugated with daunorubicin-encapsulated nanoparticles [199]. MUC1 aptamer has been used for the targeted delivery of an active metabolite of camptothecin to MUC1 positive HT29 cells [200].

Nucleolin aptamer-drug complex was taken up by breast cancer cells, effectively. Since the breast cancer cells like most of the other cancer cells, overexpress nucleolin [201].

PSMA targeted aptamer have been used to deliver docetaxel, doxorubicin, cisplatin and specifically target prostate epithelial cells [195, 202]. Targeted delivery of 5-fluorouracil (5FU) to MUC1 overexpressing colorectal adenocarcinomas [190], paclitaxel to nucleolin overexpressing breast cancer cells [203], doxorubicin to HER2-positive breast cancer cells [204] have been studied in cancer therapy. These findings indicated that aptamers, due to specificity and high affinity, can be used for efficient drugs delivery to target sites. Specific delivery of drugs is an important factor in reducing the side effects of drugs used in chemotherapy. Delivery of oligonucleotide therapeutics such as siRNAs is another mostly studied field in clinical application of aptamers [205]. Two of recently studies included CTLA-4 specific aptamer for delivery of STAT3 siRNA [206], EpCAM aptamer to deliver EpCAM siRNA [207].

Aptamers for Treatment of Disease

Aptamers can be assigned to inhibit or activate their targets; therefore, they have potentials as therapeutic agents in cancers, inflammatory diseases, infections, cardiovascular illness and metabolic diseases [208, 209]. For example, inhibition the binding of the Interleukin 17 to its specific receptor (IL-17RA) by using aptamer selected against IL-17RA, can reduce the release of inflammation mediators [210, 211]. In another study, therapeutic potential of aptamers specific for CD40 (B lymphocyte cell-surface receptor), was shown in B lymphoma and bone marrow aplasia. CD40 agonist aptamers can be used to promote bone-marrow aplasia recovery and CD40 antagonist aptamers have a direct antitumor effect on CD40-expressing B cell lymphoma [212]. Aptamers selected against the cytotoxic T cells surface proteins (CTLA-4), which send a negative signal for T cell activation, enhance tumor immunity and inhibit tumor growth [213]. Similarly, 4-1BB is a receptor on CD8⁺ T cells surface. Aptamers selected against 4-1BB have also anti-tumor effects and enhance immune response by T cells [214, 215]. Combining two or more aptamers in drug design and development, could result in an effective and selective strategy for therapeutic regimens; such as conjugation of 4-1BB aptamer with PSMA aptamer that lead to the inhibition of prostate tumor growth [209].

Aptamers can also be valuable agents in treatment of neurodegenerative diseases; for example, neurotrophin receptor, TrkB, is a cell surface protein target that its specific aptamer could be a therapeutic agent in neurodegenerative diseases [216]. Likewise, specific aptamer against Neurotensin receptor was developed, which is a type of G-protein-coupled receptors and involved in signal transduction and considered as a valuable therapeutic target [217].

Similar to the previously mentioned position of aptamers in Diabetes diagnosis, they could be applied in the treatment of diabetes complications. Diabetic neuropathy, retinopathy and nephropathy can be the result of inflammatory reactions activated by advanced glycation end products (AGEs). AGEs are a class of irreversibly cross-linked moieties as the products of sugars non-enzymatically reaction with the amino groups of proteins that initiate a complex series of rearrangements and dehydrations. In this regard, scientists designed and developed DNA aptamer directed against AGEs. Animal studies suggested that continuous administration of AGEs-aptamer improved protection against experimental diabetic nephropathy [218] and retinopathy [219].

Some of studies focused on the development of specific aptamers against anti-inflammatory factors or receptors, in order to achieve a therapeutic goal. In this way, DNA aptamers developed by special cell-SELEX method to inhibit IL-17RA-mediated synovial inflammation in an experimental mouse model of osteoarthritis [210].

The other inflammatory factors are adhesion molecules, which play critical function in inflammation process. Among them, the selectin family includes three structurally related calcium-dependent carbohydrate binding proteins, E-, P-, and L-selectin and located on the surface of endothelial cells (E- and P-selectin), platelets (P-selectin) and of leucocytes (L-selectin) [220]. In addition, Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium and their selective aptamers could limit metastasis of tumor cells [221]; along with, P-selectin specific aptamer that act as therapeutic agent for sickle cell disease [222, 223].

Anti-human L-selectin aptamers were developed and characterized as a potential therapeutic agent for intravascular targeting [224, 225]. E-selectin is another inflammatory mediator which its expression after a stimuli, is induced on the endothelial cell surface of vessels during inflammation, infections or cancers [226]. As E-selectin is absent on normal vessel, it is an attractive molecular target to develop powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. Aptamer specific to E-selectin showed selective binding to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs [227].

Moreover, Vascular endothelial growth factor (VEGF) could act as a direct pro-inflammatory mediator during the pathogenesis of rheumatoid arthritis and so its specific aptamers have potential to be a therapeutic agent of inflammatory disease [228].

Aptamers also considered as good anti-coagulant agents [229] because reversal of their effect could achieve easily by administration of a complementary DNA strand as an antidote [230, 231]. Anticoagulant aptamers are related to their activity against thrombin [232], prothrombin [233, 234], coagulation factor VII [235], von Willebrand factor (vWF) [236, 237], Factor X [238], Factor IX [239, 240]. Three of these agents entered clinical trial are ARC1779 as a an antagonist of the A1 domain of vWF and REG1 specific to the Factor IX and NU172 as a thrombin antagonist [241].

Aptamers Against Gene Regulatory Proteins

One of the valuable advantages of aptamer technology is that the aptamers could design to discriminate between much related molecules. This feature of aptamers can be very supportive to achieve selective ligands in a group of related target proteins such as gene regulatory proteins or transcription factors [242, 243]. Aptamer selection against proteins that involved in gene regulation is performed more easily, as the targets have potential of binding to nucleic acids, intrinsically. Although, most of the SELEX studies have been done for characterization of transcriptional factors’ binding sites rather than aptamer selection [244–246]. On the other hand, there are studies which select specific aptamers against transcription factors, for example, the researcher developed high affinity selective aptamers for different components of AP-1 family of transcriptional activators [247]; Or aptamers developed to human cyclin T1 to prevent human immunodeficiency virus (HIV) transcription in the human cells [248]. In addition, there are various studies of aptamer technology to design riboswitches. Artificial riboswitches contain a part of aptamer that their function controlled by binding of an external elements such as peptides or proteins and a catalytic sequence that act enzymatically [249].

Detection of Pathogenic Proteins and Toxins

Introduction of aptamer technology to the infective pathogen diagnosis and treatment have been considered as an interesting field for researchers. It is possible to select pathogen specific aptamer using whole organism or a specific pathogenic subunit such as cell surface receptors, virulence antigens or their toxins. Most toxins of infective organisms have protein structure that their related specific aptamers have been developed and characterized (Table 4).

Table 4 Aptamers selected against pathogenic proteins and toxins:

Beside toxins, pathogens’ surface antigens were also considered in aptamer design; such as aptamers selected against surface antigen of Listeria monocytogenes, one of the major food-borne pathogens, by whole cell-SELEX process [250, 251].

For the well-known pathogenic antigens, it will be possible to extract the protein purely from the whole cell organism and accordingly, design and development of aptamer could be performed as an intact protein target. For example, recombinant hemagglutinin (rHA) protein was used as target for aptamer selection and the designed aptamers exhibited the ability to bind multiple influenza A virus subtypes, H5N1, H1N1 and H3N2 [252]. Furthermore, HIV-1 is one of the most interesting target for researchers to develop specific and selective aptamers. The virus important antigens [253, 254] or structural genomic domains [255] have been considered to design of effective affinity ligands.

Aptamers Instead of Antibodies and Aptamers Specific to Antibodies

Studies of aptamer in combination with antibodies for improving their binding or detection power of both ligands have been run by different strategies; for example antibody-aptamer pincers (AAPs) was designed for thrombin and human epidermal growth factor 2 (HER2) with the aim of detection and drug delivery, respectively [270]. Aptamers and antibodies in sandwich like assays could collaborate to enhanced analysis efficiency; sometimes aptamers were used instead of primary antibodies such as that was applied in glycated hemoglobin (HbA1C) measurement [271], thrombin detection [272, 273] or C-reactive protein [274]; And sometimes applied as secondary signaling agent by affinity to the primary antibodies [275]. There have been several studies on aptamer selection against antibodies such as a high-affinity DNA aptamer that targeting F(ab′)2 fragments of saxitoxin (STX) antibodies [276], aptamer against anti-toxoplasma IgG [277] or against C595 which is an anti-MUC1 IgG3 monoclonal antibody [278].

Applications that considered for the antibody specific aptamers contained Aptamer-Apheresis Column that could specifically clear blood from β1-receptor autoantibodies in patients with cardiomyopathy [279]. The antibody specific aptamer can be used in a protein purification system with affinity column containing M2 antibodies. Scientist designed a ssDNA aptamer that blocks the function of the anti-FLAG M2 antibody. Flag peptide is a fusion peptide for recombinant protein production and purification. The aptamer competed for binding to the M2 antibody. Consequently, it resulted in the elution of Flag-tagged proteins from an immobilized M2 antibody at protein purification column [280]. In addition, this group of aptamers could be applied for highly sensitive and label-free detection of antigen–antibody reactions using optical aptamer based nanostructured sensor by the aid of an aptamer specific to constant part (Fc) of IgG [281]. The aptamers that specific to the autoantibodies would also have important value for detection and therapeutic approaches in rheumatoid diseases, like aptamers against monoclonal G6-9 anti-DNA autoantibody [282].

Conclusion

As the proteins involved in diverse biological, physiological and pathological functions, their selective ligands might introduce various functionality in basic research and practical application. Protein specific aptamers are interesting tools in this regard. They are designed and developed to replace monoclonal antibodies, improve analytical or clinical applications or introduce a novel feature of usage. Aptamers are small size affinity ligands with or without functionality, which could be stated against different domains of the target protein (apatopes). Generally, Aptamers are non-immunogenic, easily in vivo controllable, easily chemically modifiable, small in size, compatible to various applications, with easy production and uniformity in production, good stability, high affinity and selective binding. In addition, following the covalently binding at their 3′ ends to various proteins, enhance protein stability and introduce longer in vivo lifetime.

As mentioned, invention of aptamer technology could help researchers in different fields particularly, peptide and protein studies. Aptamers in therapeutic and diagnostic area are selective and stable agents that could also be improved to achieve desired pharmacokinetic properties and in the field of research, they are more easily controllable and less expensive than other selective affinity ligands. Therefore, aptamers play significant role in drug design and discovery, diagnostic strategies, analytical systems development and basic research.

About the 70 % of published research in the field of protein specific aptamer are related to analytical biosensor design. It might be the result of the best compatibility of aptamers to the nanostructures, fluorescence, electrochemical, label free sensors and high-resolution technologies such as surface plasmon resonance, resonance energy transfer systems. Aptamers also, easily manipulated to acheive the desired properties. In consequence, they represent a valuable tool in biological research.

Aptamer application for in vivo tracking, imaging, activating or inhibiting of the target proteins is another interesting point of view in clinical research and beside the one approved therapeutic aptamer (pegaptanib, brand name Macugen) for age related macular degenerative disease, there are some other therapeutic and diagnostic aptameric agents in different stages of clinical trials. In general, aptamers are the promising artificial biological elements with broad ranges of application especially in the world of proteins.