Aptamers are oligonucleotides derived from an In vitro evolution process called SELEX. Now, with the development of SELEX process, the isolation of small oligonucleotides that possess the capacity to recognize various classes of target molecules have been made much easier. Aptamers have been evolved to bind proteins which are associated with a number of disease states. They are oligonucleotides sequences that have the tendency to bind to a wide range of target molecules, such as drugs, proteins or other organic or inorganic molecules with high affinity and specificity. These molecules offer a tough rivalry to the antibody classes in terms of therapeutic and diagnostic applications. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these associated advantages aptamers are not used on a massive scale commercially, the aptamer-based diagnostic research is still in its infancy. With the increasing use of aptamers in numerous diagnostics, therapeutics and other such aspects, it is likely that the perception of nucleic acid therapeutics will be changed and that aptamers may form the basis of future therapeutics in aquaculture research.

Keywords:  Aptamers, SELEX technique, Antibodies, DNA, RNA, Yeast, Trypanosome, oligonucleotides, Temperature, Picomolar range, Dyes, Amino acids, Antibiotics, Peptides, Protein, Vitamins

SELEX, Systematic Evolution of Ligands by Exponential Enrichment; NMR, Nuclear Magnetic Resonance; VHSV, Viral Haemorrhagic Septicaemia Virus; OMPs, Outer Membrane Proteins; LPS, Lipopolysaccharides

“Aptamers” is derived from the Latin word “aptus”, meaning “to fit”. Aptamers are oligonucleotides (DNA or RNA) that have tendency to bind with high affinity and specificity to a wide range of target molecules, viz. drugs, proteins or other organic or inorganic molecules.¹ whole viruses and bacteria and even multicellular organisms such as yeast and trypanosome .² It offers the opportunity of developing affinity-based detection systems.

Antibodies, providing molecular recognition cater a wide range of applications. They have made wide contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are routinely used in clinics. Development of the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process however has made possible, the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

Aptamers are generated from a library of nucleotides or proteins containing approximately more than 1015 different molecules and are selected In vitro by SELEX process.³ Great efforts have been made to make aptamers clinically relevant for detection of diseases like viral disease (cancer, HIV) and macular degeneration. Aptamers are highly specific, relatively small sized, non-immunogenic oligonucleotides that have molecular recognition properties and are advantageous over antibodies .⁴

1. DNA or RNA Aptamers: short strands of oligonucleotides.
2. Peptide Aptamers: short variable peptide domain, attached at both ends to a protein scaffold.

Aptamers are single-stranded nucleic acids with defined tertiary structures which selectively bind to target molecules. Aptamers also possess ability to bind a complementary DNA sequence to form a duplex structure. The Duplex is formed between a fluorophore labelled DNA aptamers and a small oligonucleotide which is modified with a quenching moiety (denoted QDNA). When the target is absent, the aptamers bind to QDNA, then after it brings the fluorophore and the quencher into close proximity for maximum fluorescence quenching. When the target is introduced, the aptamers prefer to form the aptamer-target complex. The switching of the binding partners for the aptamers occurs in conjunction with the generation of a strong fluorescence signal owing to the dissociation of QDNA. Aptamers are capable to form stable three-dimensional structures in aqueous solution. Single-stranded oligonucleotides follow the same rule of complementarity of nucleic acid bases as do double-stranded molecules, thus it is relatively simple to predict their secondary structure. Different types of secondary motifs of Aptamers are (a) Hairpin (b) Pseudoknot (c) G-quadruplex (d) Three-dimensional representation of an anti-malachite green aptamer in complex with its ligand. The process of folding structures in the solution and the ligand-induced conformational changes were studied to be strongly dependent in the presence of divalent cations. Structures can be experimentally determined by crystallography.⁵ or Nuclear Magnetic Resonance (NMR) spectroscopy (Figure-1).

Figure 1 Aptamers secondary structure.²⁰

Development of aptamer by SELEX technique enablesthe selection of specific oligonucleotides from library of randomized molecules. SELEX method has characteristics to identify the unique RNA/DNA molecules, from very large populations of random sequence oligomers (DNA or RNA libraries). Aptamers possess a randomized region of 30–50 nucleotide flanked by constant sequences which helps in PCR amplification.The criteria of SELEX is to stimulate evolution (Systematic Evolution of Ligands). It can be done with both ways by introducing many rounds of selection (usually 10–15) and by exponential amplification of ligands via PCR after each round (Exponential Enrichment). Finally, the DNA strands are separated and those corresponding to the original library strands are subjected to a next round of selection.² The manipulation of selection parameters (pH, temperature or buffer composition) can be easily done to obtain optimal aptamers. At the initial step, incubation of DNA/RNA sequences from library is done with an immobilized target. Unbounded DNA/RNA molecules are washed away and the ligands that get binded are eluted and amplified, either by the process of standard PCRor RT-PCR in the case of RNA aptamers. On the completion of several rounds, the selected molecules are randomly cloned and sequenced. Aptamers have a very high affinity to their targets, with dissociation constants varies from the micromolar to low picomolar range, comparable to those of some monoclonal antibodies, sometimes even better. Single-stranded DNAs also fold In vitro, structures forming like stem-loop, internal loops, etc., even if less stable than the corresponding RNA structures.⁶ High-affinity aptamers after being selected bind to hundred different target molecules such as organic dyes.⁷ amino acids.⁸ antibiotics .⁹ peptides ¹⁰ proteins .¹¹ and vitamins .¹² (Figure- 2).

Figure 2 Nucleic acid aptamers: clinical applications and promising new horizons .⁴

In the case of RNA aptamers, they have to be first reverse-transcribed thus an RNA polymerase promoter (usually T7) must be introduced during library construction.² (Figure-3).

Figure 3 Analytical applications of aptamers ²¹

High affinity property of aptamers for their targets can be seen by their capability of folding upon binding with their target molecule - they can either incorporate small molecules into their nucleic acid structure or integrate into the structure of larger molecules such as proteins. Aptamer receptors are more advantageous as compared to the antibodies which make them very promising in analytical and diagnostic applications. Most of the antibody production in biological systems by inducing an immune response to the target analyte, but the immune response can be failed also if the target molecule, i.e. protein, has a similar structure to endogenous proteins. But isolation of aptamers by In vitro methods are independent of animals. Generation of antibodies in host means that is the animal immune system is involved which selects the site on the target protein to which the antibodies bind Whereas aptamer selection process can be manipulated to generate aptamers that bind a specific region of the target and which has specific binding properties in different binding conditions. After selection of aptamer is done, aptamers are produced by chemical synthesis and modifications in the aptamer can be achieved by enhancing the stability, affinity and specificity of the molecules. However, the primary limitation on the use of aptamers (mainly RNA aptamers) as recognition elements has been their nuclease sensitivity which is very critical for their use in ex vivo and In vivo applications.¹³ By chemical modification of the Ribose ring at the 2-position, the stability of such molecules can be improved. In general, RNA aptamers spossess the possibility of intracellular expression, whereas DNA aptamers are more stable. No significant differences in specificity or binding properties have been observed between these two types.

Food safety control

Food safety plays a very important role in our day-to-day lives because varieties of toxic and harmful substances in food affect human health. Aptamers technique is most promising, which helps to overcome many disadvantages of other existing detection methods in food safety, such as long detection time, low sensitivity, difficult, and expensive antibody preparation. Food borne diseases represent a global public health challenge. Each year, millions of people get sick, and some even die as a result of the ingestion of unsafe food and water. The so-called globalization of the food production, processing and distribution, further complicates the problem. Because of the increase in consumers concerns about what is in their food and its relation with health, control over the safety and quality of food has become tighter, particularly in developed countries where food availability problems are much lower.

Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfil these characteristics, and thus have surfaced as promising alternatives to natural receptors. Therefore, aptamers can be used for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety.

Cellular biomarker assay

Cell-SELEX enabled isolation of many aptamers, which recognizes particular cell types. This include range from bacteria through unicellular parasites to mammalian cells.

Medical diagnostics

The perfect sensor used for medical diagnostics ought to be highly specific, sensitive, repetitive, fast and easy to read. It should also be non-reactive and perform well under physiological conditions. Aptamers meet all these requirements. Due to the wide spectrum of potential aptamer targets, detection of virtually all toxins, drugs and metabolites in body fluids at exceptionally low concentrations becomes feasible.

Electrochemical sensor

Sensors possess higher sensitivity and cost of detection is low as no sophisticated optical machinery is required. Other important application of aptamers are as followings:

1. Detection of biological terrorist threat agents.
2. Application to the detection of food borne pathogens.
3. Aptamers may also used as probes to view tumour cells and might prove very helpful in cancer therapy.
4. New drug development, drug delivery, bio-imaging hazard detection, food detection (Table 1).

Successful application of aptamer in fisheries

In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder. Viral haemorrhagic septicaemia virus (VHSV) is a serious disease which has impact on wild and cultured. Hence, a most powerful therapeutic method against VHSV infection is needed to be developed. Aptamer is a novel technology and promising method for diagnostics and therapeutics. Here, it is aimed at selecting RNA aptamers that specifically binds to and inhibits the growth of a strain of fish VHSV both In vitro and In vivo .¹⁴

Various Aptamers screening technologies summarizes the recent Salmonellae are a significant causative organism of food borne illness worldwide. Selection and evaluation of DNA aptamers were done for the capture and detection of Salmonella enterica serovar typhimurium. Aptamer sequences were modified against S. typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS) .¹⁵

Future prospects

The virtually infinite array of potential target molecules places aptamers among the most powerful tools in biotechnology. Aptamers possess the potential to improve many of the standard laboratory techniques, which involves purification, detection or concentration measurement of a molecule of choice. Aptamers that have reached the clinical trials drugs can be developed against diseases such as, neo-vascular age-related macular degeneration, diabetic retinopathy, haemophilia, acute myeloid leukaemia, non-small-cell lung cancer, renal cell carcinoma or an acute coronary syndrome. Apart from having enormous applications, aptamers have not yet been used in aquatic animals. The reason is that there are some limitations that have to be considered for In-vivo applications, such as aptamer stabilization, delivery method and mass production .¹⁶  But now several methods have been used to enhance the stability of RNA aptamers, such as amino or fluoro modifications at the 2’ position of pyrimidine.^17-21 These results may lead to a new method for improving the health of aquatic animals.

None.

None.

1. Jayasena SD Aptamers: an Emerging Class of Molecules that Rival antibodies in Diagnostics. Clin Chem. 1999;45(9):1628–1650.
2. Stoltenburg R, Reinemann C, Strehlitz B SELEX––a (r)evolutionary method to generate high–affinity nucleic acid ligands. Biomol Eng. 2007;24(4):381–403.
3. Tuerk C, Gold L  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 2007;249(4968):505–510.
4. Ni X, Castanares M, Mukherjee A, et al. Nucleic acid aptamers: clinical applications and promising new horizons. Curr Med Chem. 18(27):4206–4214.
5. Padmanabhan K, Padmanabhan KP, Ferrara JD, et al. The structure of alpha–thrombin inhibited by a 15–mer single–stranded DNA aptamer. J Biol Chem. 1993;268(24):17651–17654.
6. Harada K, Frankel AD Identification of two novel arginine binding DNAs. EMBO J. 1995;14(23):5798–5811.
7. Ellington AD, Szostak JW ( Selection in vitro of single–stranded DNA molecules that fold into specific ligand–binding structures. Nature. 1995;355(6363) 850–852.
8. Michael Famulok, Jack WS Stereospecific recognition of tryptophan agarose by in vitro selected RNA. J Am Chem Soc. 1992;114(10):3990–3991.
9. Schürer H, Stembera K, Knoll D, et al. (2001) Aptamers that bind to the antibiotic moenomycin A. Bioorg Med Chem. 1992;9(10):2557–2563.
10. Baskerville S, Maria Z, Andrew DE Anti–Rex aptamers as mimics of the Rex–binding element. J Virol. 1999;73(6):4962–4971.
11. Klug SJ, Hüttenhofer A, Famulok M  In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA–binding sites, reveals one major interaction domain at the carboxyl terminus. RNA. 1999;5(9):1180–1190.
12. Wilson C, Nix J, Szostak J Functional requirements for specific ligand recognition by a biotin–binding RNA pseudoknot. Biochemistry. 1998;37(41):14410–14419.
13. Famulok M, Mayer G, Blind M Nucleic acid aptamers–from selection In vitro to application In vivo. Acc Chem Res. 1998;33(9):591–599.
14. Punnarak P, Santos MD, Hwang SD, et al. RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV). Mar Biotechnol (NY). 2012;14(6):752–761.
15. Joshi R, Janagama H, Dwivedi HP, et al. Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars. Mol Cell Probes. 2009;23(1):20–28.
16. Osborne SE, Matsumura I, Ellington AD Aptamers as therapeutic and diagnostic reagents: problems and prospects. Curr Opin Chem Biol. 1997;1(1):5–9.
17. Ruckman J, Green LS, Beeson J, et al. 2′–Fluoropyrimidine RNA–based aptamers to the 165–amino acid form of vascular growth factor (VEGF165). Inhibition of receptor binding and VEGF–induced vascular permeability through interactions requiring the exon 7–encoded domain. J Biol Chem. 1998;273(32):20556–20567.
18. Famulok M, Hartig JS, Mayer G Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev. 2007;107(9):3715–3743.
19. Luzi E, Minunni M, Tombelli S, New trends in affinity sensing: aptamers for ligand binding. TrAC Trends in Analytical Chemistry. 2003;22(11):810–818.
20. Radom F, Jurek PM, Mazurek MP, Aptamers: molecules of great potential. Biotechnol Adv. 2013;31(8):1260–1274.
21. Tombelli S, Minunni M, Mascini M Aptamers–based assays for diagnostics, environmental and food analysis. Biomol Eng. 2007;24(2):191–200.