Comparison between Monoclonal antibody vs. Aptamer:
Aptamers are a short polymer of oligonucleotides (natural or modified) that can bind to its cognate target (small molecules to large macromolecules like proteins, cells, microorganisms etc.) with high affinity and selectivity. They can fold into unique secondary and tertiary conformation in solution (pH, ionic concentration) and bind to their targets in a specific manner (binding constants in sub-nano to pico molar range). They rival the monoclonal antibodies and other specific biological ligands with respect to affinity, stability, robustness, non-immunogenicity and facile to synthesis. Nucleic acid aptamers are selected from an oligonucleotide library by an iterative process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment Analysis). These aptamers are compatible to any kind of chemical modification, conjugation and functionalization. Briefly, this chapter discusses about the diagnostic and therapeutic application of aptamers.
- chemical antibodies
Aptamers (Latin word
Compared to antibodies, aptamers can be produced using cell-free chemical synthesis and are therefore less expensive for large-scale manufacture. Aptamers exhibit extremely low variability between batches and have better controlled post-production modification, they are minimally immunogenic, and are small in size. (Table 1) The rapidly growing aptamer industry was predicted to reach US $244.93 million by 2020.  Presently more than 40 companies are actively engaged in diagnostics and therapeutics research to commercialize these “magic bullets” globally (EU countries, Asia, USA, UK etc.).  The largest company is “SomaLogic” (company based on SOMAmer- a patented “Slow Off-rate Modified Aptamer) founded by Prof. Larry Gold at Colorado, USA. Since the advent of aptamers scientists and researchers exploit different applications of aptamers that reflects the following trends in the publications. (Figures 1 and 2).
2. Basics of SELEX screening process
Back in 1990, two individual groups Prof. Larry Gold and Craig Tuerk from University of Boulder, USA and Prof. Jack Szostak and his student A.D. Ellington from Havard University, USA discovered the evolution process to obtain the oligonucleotide binders and they coined the term ‘Aptamer’ and the process as ‘SELEX’. [24, 25] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a common screening process by which aptamers can be selected from an aptamer library which consist of 1024–25 number of various sequences. The method attempts to isolate an aptamer of interest from a pool of randomized library by an iterative cycle of incubation with the target, partitioning and amplification, until the pool of aptamers enriched enough to fit with the target. The SELEX procedure iterates over five basic steps- incubation of aptamer pools with the target, binding, partitioning and washing (to get rid of non-binders which are loosely bound with the target), then elution of positive target-bound aptamers and amplification of enriched pools. Traditionally, the positive pool eluted from last round is being analysed, and high-throughput sequencing is performed.
An array of different RNA and DNA aptamers were isolated against a vast array of targets: ions,  low molecular weight metabolites, [27, 28] proteins, [29, 30, 31] sugar moieties  lipids,  and even whole cells. [34, 35]
3. Library selection
To select highly selective, specific aptamers, design of the initial aptamer library is the first and foremost step. In case of determination of the length of the random region researchers should consider the sequence space and structural diversity. The complexity of the initial aptamer library depends on the length of the random window of the aptamer library (If the random window is 40 and if we consider DNA aptamer library, so the complexity of the library is: 4^40 that equals to 1024–25). 
Special libraries would consist of specifically designed flanking sequences directing the aptamers to form a specific secondary structure, or include modified nucleotides. In capture SELEX, there is unique docking sequence (12–14 nucleotides long) which enables the library in such a way, that highly sensitive aptamers can be fished out against small molecules. [37, 38] The extended genetic alphabets or combination of artificial xeno nucleic acids (XNA) greatly broaden the diversity of sequences and can influence the properties of the aptamers, such as their in vivo stability or nuclease resistance. [39, 40, 41, 42] Modified nucleotides can be introduced either during the library synthesis or in the post-selection optimization.
In a review article, Maria
4. Various SELEX processes
Specific aptamers are screened by the iterative processs of SELEX from a highly diverse pool of oligonucleotides. [44, 45, 46] After the incubation of the random aptamer pool with the target followed by the removal of non- binding aptamers, the bound aptamer species are recovered. These recovered nucleic acid sequences are amplified with PCR (in the case of DNA aptamer) or RT-PCR (for RNA aptamers). In addition to selection against a purified target molecule, SELEX process can be performed against live bacterial cells and even in mammalian cell lines to isolate cancer cell specific aptamers and furthermore it can lead to the identification of novel biomarkers. [47, 48]
A giant advancement of SELEX technology has been made since its discovery in 1990. Conventional SELEX is a well-established and effective method but due to its immense time- and labor-consumption, continuous improvement of alternative methods for aptamer selection has been inevitable.
High throughput SELEX (HT-SELEX), Functional screening (Microfludics or Flow cytometry based SELEX), Cross-over SELEX (where the target is alternatively changing from proteins and cells), (Figure 3)
5. Modifications of naturally occuring aptamers
DNA is the backbone of central dogma of our life cycle. Moreover, any form of nucleic acids play a crucial role in our genetic codon. DNA/RNA is an essential bio-macromolecule consist of nucleotide bases such as adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C).
There are various types of modifications (nucleotide base modifications, phosphate backbone modifications, peptide mimic oligonucleotides PNA etc.) available which can prevent aptamers from nuclease degradation. Locked nucleic acid (LNA) is one among them where 2′-oxygen has been linked to the 4′-carbon of the ribose sugar by a methylene bridge, thus completely locking the sugar into a 3′-endo conformation. LNAs increase the thermodynamic stability, binding affinity, and enable the oligonucleotides to prevent serum degradation. [50, 51, 52] These modifications enable the aptamers for biological applications.
Compared to LNAs, the unlocked nucleic acid (UNA) is an acyclic ribose derivative that has increased flexibility. UNAs do not consist the C2’-C3’ bond, which confers the flexibility observed in this modified nucleotide.  LNAs increase the melting temperature of the nucleotide by 1–10°C per LNA insertion but UNAs reduce the melting temperature by 5–10°C retaining the nuclear resistance. In case of, Peptide nucleic acid (PNA) in which sugar-phosphate backbone is modified by short stretch of N-(2-aminoethyl)-glycine units connected by peptide bonds, enhances biostability of the modified candidates. 
5.1 Aptamers in Drug development pipeline
Aptamers have been incorporated in drug development pipeline as they have the capacity to block the downstream signalling (phosphorylation of kinases etc.) of different biomolecules. They can play an important role to regulate various cellular crosstalks. To screen therapeutic aptamers either DNA aptamers or 2′-fluoro modified RNA, a combination of 2′-fluoro pyrimidines and 2′-hydroxyl purines (fYrR) are of major interest. fYrR is the “nuclease stable RNA” and can be easily generated by Y639F modified T7 RNA polymerase. Fovista, an anti-platelet derived growth factor (PDGF) aptamer, was previously DNA aptamer but later modified to augment the stability with the addition of backbone modifications.  As with the 2′-fluoro modification, the 2’-OMe modifications adopt a C3’-endo conformation. US FDA approved the first aptamer (Macugen®, pegaptanib sodium) in 2004 against vascular endothelial growth factor for the treatment of age-related macular degeneration.  This aptamer was modified with 2′-fluoro-pyrimidines and 2’-O-methyl-purines. The stability of the small aptamer was a critical factor but later which can be circumvented with a 3′-cap and a polyethylene glycol molecule, the half-life of Macugen® was extended to 131 hours at max. [57, 58] Anti-vascular endothelial growth factor (VEGF 165) aptamer Macugen, and an anti-Factor IXa aptamer REG1 were both selected from fYrR libraries, and subsequently 2′-O-methyl nucleosides have been incorporated in order to increase serum stability. 
There is a plethora of polymerase enzymes like KOD, Pwo, Phusion, Superscript III, vent (exo-), T7 polymerase have all been shown to be capable of incorporating modified triphosphates into DNA and RNA strands, which open up a new opportunities in aptamer selection strategies.  The use of Pfx DNA polymerase allows amplification of Ds-Px base pair in Ex-SELEX protocol where extended genetic alphabets were included in complexity of nucleic acid library. 
Several limitations of aptamers should be considered in the process of
In the field of oncology, two aptamers, namely, AS1411 and NOX-A12, have entered clinical trials. [45, 64] AS1411 (formerly ARGO100; Antisoma) is a guanine quadruplex aptamer obtained from a guanine-rich oligonucleotide library in the anti-proliferation screen, which is not a typical SELEX process.  The guanine quadruplex structure benefits AS1411 because it is resistant to nuclease degradation and enhances cell uptake. In
6. Aptamers as diagnostic agents
Aptamers can be used
Radiohalogens (fluorine-18, bromine-76, iodine-125 etc.) are the most commonly used for radiolabelling oloigonuclotides which are often accompanied with prosthetic groups. Recently, click-chemistry for radiofluorination was demonstrated on antisense oligonucleotides and siRNAs. [74, 75] Another report used photoconjugation as strategy for the radiofluorination of an aptamer.  Oligonucleotides have also been radiolabeled with the radiohalogens such as bromine-76 for PET imaging and iodine-123 for SPECT (Single photon emission computed tomography) imaging. In addition, iodine-125 has been used to radiolabel antisense oligonucleotides, aptamers and spiegelmers for theranostic applications. Due to the harsh and non-aqueous reaction conditions usually needed to radiolabel prosthetic groups, it is performed before the conjugation process to the oligonucleotide. 
Till date, a plethora of aptamers have been modified or labelled with radioactive molecules. Aptamers against several important biomarkers like PMSA, Tenascin C, thrombin, MUC1 were already exploited for radiolabelling. Aptamer-based radiopharmaceuticals were primarily developed for imaging and therapy of cancer diseases, metabolic disorders and others. The aptamers are mainly radiolabeled with technetium-99 m for SPECT (Single photon emission computed tomography), PET (Positron emission tomography) imaging. Very few aptamers were published related to PET imaging, and there is only one study of radiolabeled aptamers for therapy by Bandekar
Molecular nuclear imaging technique is a diagnostic process of non-invasive visualization of any disease
The first radiolabeled aptamer for nuclear imaging was discovered by Charlton et al. A DNA aptamer, NX21909, was selected against human neutrophil elastase, an enzyme which is secreted by neutrophils and macrophages during inflammation to kill pathogens. 
Aptamer TTA1, an RNA aptamer targeting the extracellular matrix protein tenascin C (TN-C), was the first radiolabeled aptamer which was used as molecular cancer imaging agent. Aptamer TTA1 was generated by a cross-over SELEX involving the purified recombinant TN-C protein and TN-C-positive U251 glioblastoma cells. [79, 80]
7. Lightup aptasensors for diagnostic applications
There are a unique group of aptamers (generally RNA aptamers) which can bind specifically with their cognate fluorogen molecules like DFHBI, thiazole orange, thioflavin T etc. [49, 81, 82] The non-fluorescent moelcules (native unbound state) become fluorescent (bound state) after binding to the aptamers and these “light-up” aptamers generate fluorescence signal. In the omni-presence of target molecules (small pre-miRNAs) and malachite green (fluorogen) light up aptasensors ‘malaswitch’ exhibit fluorescence enhancement.  We can engineer the small-molecule specific aptamers (like aptamers for some pesticides, toxins, small metaboltes) in such a way, that combined with light-up aptamers, they can generate a detectable signal. Light up aptasensors are promising alternative biosensor for label free sensitive detection of small molecules. 
8. Future perspectives
With more focus on