Oligonucleotide Based Therapeutics:  Aptamers


Bhosale AV, Hardikar SR*, Patil Naresh , Bhujbal PU, Khirsagar AA and Malvankar SR

PDEA'S SGRS College of Pharmacy ,Saswad ,Pune 412301 (MS) India

*Corresponding Author E-mail: srhsgrs@yahoo.co.in



The development of systematic evolution of ligand by exponential enrichment (SELEX) process, made possible the isolation of oligonucleotide sequence with the capacity to recognize virtually any class of target molecules with high affinity and specificity .These oligonucleotide sequences referred as ‘Aptamers’ and have wide diagnostic ,therapeutic and analytical applications. Pre and post SELEX modifications confer biological stability to aptamers against nucleases. This enhances molecular diversity of oligonucleotide libraries and further enhances probability of finding aptamer with unique properties. The differentiating feature of an aptamer from other nucleic acid sequences (antisense nucleotide, si RNA etc) is its ability to fold into a tertiary structure to create a binding pocket to precisely and specifically interact with target.




Systemic Evolution of Ligands by Exponential  Enrichment is a combinatorial technique in molecular biology for producing Oligonucleotides of either single stranded DNA or RNA that specifically bind to a target ligand are called as Aptamers..1, 2.


The process begins with synthesis of a vary large oligonucleotide library consisting of random generated sequences of fixed length flanked by constant 5’ and 3’ ends that serves as primers. For a randomly generated region of length n, the number of possible sequences in the library is 4n .The sequences in the library are exposed to the target ligand which may be a protein or a small organic compound. The sequences that do not bind the target even weakly are removed, usually by affinity chromatography. The bound sequences are eluted and amplified by RT-PCR to prepare for subsequent rounds of selection on which the stringency of the elution conditions is increased to identify the highest binding sequences .3, 4


The molecular weight of aptamers is 10–15 kDa; hence aptamers are expected to have rapid tissue and tumor penetration, as well as fast blood clearance. Aptamers are chemically stable to all but the harshest environmental conditions and can be boiled or frozen without loss of activity.  They may be produced on bench top using standard molecular biological techniques or they may be chemically synthesized at micrograms to kilograms scales. As synthetic molecules they are amenable to a nearly infinite variety of modifications designed to optimize their properties for a specific application.


They may be circularized, linked together in pairs, or clustered onto the surface of a flat globule. For in vivo applications, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes (nucleases). Other chemical appendages can alter their biodistribution or plasma residence time following intravenous administration. Variety of aptamers have now been evolved  of extremely high binding affinity of target ligands, including small molecules such as ATP and adenosine , proteins such as prions and  vascular endothelial growth factor (VEGF).5-10 


Since the discovery of the aptamers, many of us have wondered why nature did not make use of such a claver mechanism. Now it was discovered that this mechanism is indeed used by nature. Metabolite binding domains in mRNA’s, which refold after ligand binding, were recently found by cyanocobalamin, thiamine and FMN 11. These recent findings give us a taste of what is waiting to be discovered and clearly show that metabolite-RNA complexes will be used in the future for a yet unpredictable number of applications.2


Oligonucleotide Based Therapeutics: Aptamers:-

Aptamers are single stranded macromolecules composed of nucleic acid, such as DNA or RNA, that bind tightly to a specific molecular target. Like all nucleic acids, a particular aptamers may be described on paper by a linear sequence of nucleotides (A, U or T, CandG) typically 15-60 letters long. Aptamers can fold into a three dimensional structure  based on their nucleic acid sequence to bind with the target.4 The shape of the Aptamers allow it to bind tightly against the surface of its target molecule. Many of the in vitro selected Aptamers adopt their final fold only after ligand binding, with the ligand being the essential part of the structure.







Aptamers as ligands in chromatography and electro chromatography For analyte capture and separation

1.    Immobilized DNA Aptamers for human L-selectin (for purification)

2.    Thrombin binding DNA aptamer as a protein capture system (for separation)


Target specific chiral separation

Anti tyrosine specific L-RNA Aptamers for separation of enantiomers of tyrosine and analogues.


Detection of Biomolecule by fluorescence labeled Aptamers

Tat protein of HIV-1


In the absence of the ligand it is rather unstructured. The term Aptamers derived from the Latin  word ‘Aptos’  means ‘ to fit’ and was chosen to emphasize this lock and key relationship between Aptamers and their binding partners. Aptamers recognize their targets with extraordinary affinity and specificity. (See Fig. No.1)


The surface area of interaction between an Aptamer and its molecular target is relatively large, so even small changes in the target molecule can disrupt aptamer association. Thus aptamers can distinguish between closely related but nonidentical members of a protein family, or between different functional or conformational states of the same protein. In a striking example of specificity, an aptamer to the small molecule theophylline (1, 3-dimethyl xanthine) binds with 10,000 fold lower affinity to caffeine (1,3,7-trimethyl xanthine) that differs from theophylline by a single methyl group.  


With the development of in vitro selection procedure, it became possible to isolate RNA Aptamers for probably every water soluble ligand and the small size of these Aptamers made them perfect tools to explore the rules that govern recognitions of small molecules by RNA. To date, there has not been a restriction on the type of target for which high affinity Aptamers could be identified.13, 14


Aptamer   identification:-

The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is the technique for screening very large combinatorial libraries of oligonucleotides by an iterative process of in vitro selection and amplification. It is also referred as in vivo selection or in vitro evolution and is a combinatorial technique in molecular biology for producing oligonucleotides of either single stranded DNA or RNA that specifically bind to target ligand.2 The process begins with the synthesis of a very large oligonucleotide library consisting of randomly generated sequences of fixed length flaked by constant 5’ and 3’ ends that serve as primers. Theoretically, for a randomly generated region of length n, number of possible sequence in the library is 4n. However in practice the complexity of a typical combinatorial oligonucleotide library obtained from 1µmol scale solid phase DNA synthesis is limited to 1014 to 1015 individual sequences. The success of finding unique and rare molecule that interact with a target parallels the diversity of the libraries used. The degree of molecular diversity present in the random sequence oligonucleotide libraries supersedes that of other combinatorial libraries used for screening.13

The sequences in the library are exposed to the target ligand which may be a protein or small organic compound and those that do not bind the target even weakly are removed by any one of the physical separation techniques, usually by affinity chromatography, immuno precipitation, electrophoretic mobility shift or filter binding techniques.14 Amplification of selected oligonucleotide by PCR  provides a new pool, enriched in sequences with improved affinity for the target relative to the starting pool. Successive iteration of the selection and amplification cycles evolved a product pool containing only those oligonucleotides (Aptamers) having a greatest affinity.4 Once affinity saturation is achieved after several rounds of selection / amplification, the enriched library is cloned and sequenced to obtain the sequence information of each member. Individual sequences are further characterized on the basis of their ability to bind to the target. (See Fig. No.2)


The number of cycles required for Aptamers identification is usually dependent on the degree of stringency imposed at each round as well as on the nature of target. For the most targets, affinity enrichment is reached within 8-15 cycles. Aptamers that come out of a SELEX experiment are full length sequences containing the fixed sequences that were included to aid the amplification process. These full length Aptamers are generally 70-80 nucleotides long and could be truncated to eliminate nucleotide stretches that are not important for direct iterations with the target or for folding into the structure that facilitates target binding . The identification of truncated Aptamers restricted  to the minimal target binding domain requires some efforts , but it has  been successfully carried out to obtain functional Aptamers less than 40 nucleotides long .15-20 In the majority of cases , the fixed sequence regions in random sequence libraries used for the SELEX process , thereby producing  short aptamers sequences. Including cloning and sequencing, a typical SELEX experiment may take approximately 2-3 months.


Fig. No. 1: Aptamer


Fig. No. 2:  Process of SELEX


sequences (RNA/DNA)




In vitro

selection (SELEX)

(Ligand binding or                                                                                                               Transcribe

catalytic activity)                                                                                                                   Into RNA



                                Discard unbound                                                                                     Repeat

                                molecules                                          Amplify                                          cycle 4-10

                                                                                           (PCR)                                            times



                                         Collect bound                                                                       Clone sequence

                                            molecules                                                                                         characterize    




In vivo stability of aptamers:

In order to move Aptamers from the bench to the clinic, several hurdles have to be taken. The most prominent one are the issues of Aptamers stability in biological fluids and production costs. Since 70% of Aptamers are RNA nucleotides they are degraded by nucleases commonly present in biological fluids. Because the nucleases that are most abundant in biological fluids appear to be pyrimidine specific endonucleases, substitutions at the 2’ positions of pyrimidine nucleotides alone is sufficient to protect an RNA sequence from degradation in biological fluids.21-22 groups at the 2’ position of the sugar are substrates for the enzymes used in the SELEX process. As a result, Aptamers with enhanced survival times in biological fluids have been selected successfully from libraries containing pyrimidines modified with 2’ NH2 and 2’ F functional groups. These Aptamers resistant to nucleases Pre-SELEX modifications-Pyrimidine nucleotides substituted with amino (NH2) and fluoro (F) functional are well suited for both diagnostic and therapeutic applications. Thus stabilization of Aptamers against abundant nucleases has been improved by the introduction of chemically modified nucleic acid libraries. (See Fig. No.3) On the other hand, compared to unmodified RNA sequences, unmodified DNA sequences are generally more nuclease resistant.


In post  SELEX modifications, chiral principles are introduced in SELEX process, which rely on the substitution of RNA’s 2’ OH groups in order to generate nuclease resistant aptamers on the basis of  L RN  or L DNA, so called spigelmers (from the German ‘spigel’ meaning mirror).13,23


Spigelmers are identified through in vitro selection of an unmodified natural D RNA or D DNA library against the mirror image configuration (enantiomers) of a drug target. The selected aptamer sequences are then synthesized in their unnatural enantiomeric configuration as L RNA’s or L DNA’s. Following the rules of symmetry these spigelmers bind to the mirror image selection target. Aptamers with pre


and post SELEX modifications as well as spigelmers have been reported to be stable for many hours in biological fluids.24



Diagnostic applications:

Kohler and Milstein reported the discovery of the monoclonal antibody technology that allowed the production of a unique antibody in large quantities.. Because of the enormous practical value of antibodies, monoclonal antibody technology became very popular and was embraced by researchers in all corners of the world.  There are certain limitations, however, associated with antibodies: 25-26


1.      The antibody identification process starts within an animal; therefore, antibody generation becomes difficult with molecules that are not well tolerated by animals, such as toxins Furthermore, antibodies against molecules that are inherently less immunogenic are difficult to raise.

2.      By enlarge, the generation of hybridomas are restricted to rat and mouse, limiting the use of antibodies in therapeutic applications. Antibodies of non-human origin have implication in diagnostic applications.

3.      The identification and production of monoclonal antibodies are laborious and could become very expensive in searches for rare antibodies that require screening of large number of colonies.

4.      The production of antibodies has challenges of its own. Frozen stocks of antibody- producing cells should be stored at multiple sites to overcome accidental losses or the death of the cell lines. Typically, high yields of monoclonal antibodies are obtained by growing the hybridomas in the peritoneal cavities of animals and purifying the antibodies from ascites fluid. Some hybridomas are difficult to grow in vivo, thus restricting this route of antibody production.

5.      The performance of same antibody tends to vary from batch to batch, requiring immuno assays to be re-optimized with each new batch of antibodies.

6.      Although the production of antibodies is subject to in vivo variation, the identification of antibodies is restricted by in vivo parameters. In other words, identification of antibodies that could recognize targets under conditions other than physiological conditions is not feasible.

7.      The kinetic parameters of antibodies- target interaction can not be changed on demand.

8.      Antibodies are sensitive to temperature and undergo irreversible denaturation. They also have a limited shelf life.


Alternatively, it is possible to consider an entirely different class of molecule, aptamers, to made the   shortcomings of the antibodies. In this regard, Aptamers have the following advantages:

1.      Aptamers are identified through an in vitro process that dose not depend on animals, cells, or even in vivo conditions. As a result, the properties of Aptamers can be changed on demand.

2.      Selection conditions can be manipulated to obtain Aptamers with properties desirable for in vitro diagnostics. For example, Aptamers that bind to a target in a non physiological buffers and at non physiological temperatures could be identified. Similarly, kinetic parameters such as, the on- and off- rates of Aptamers, could be change on demand.

3.      Because animals or cells are not involved in Aptamer identification, toxins as well as molecules that do not elicit good immune responses can be use to generate high affinity aptamers

4.      Aptamers are produced by chemical synthesis with extreme accuracy and reproducibility. They are purified under denaturing conditions to a very high degree of purity. Therefore, little or no batch to batch variation is expected in aptamer production.

5.      Reporter molecule such as fluorescein and biotin can be attached to aptamers at precise location identified by the user. Functional group that allows subsequent derivatization of Aptamers with other molecules can also be attached during chemical synthesis of aptamers.

6.      Aptamers undergo denaturation, but the process is reversible. Once denatured, functional Aptamers can be regenerated easily within minutes. They are stable to long term storage and can be transported at ambient temperature.


Over the past 3 decades antibodies have been the reagent of choice for the diagnostic assays. Consequently diagnostic platforms that are commonly used today were evolved to better suit antibodies. The discovery of Aptamers whose affinity and specificity parallel to those of antibodies is expected to have a future impact on diagnostics. Now a days aptamers that have been tested in different diagnostic platforms show very encouraging results. The value of Aptamers in most cases will be in applications or format where the performance of antibodies is inadequate rather than replacement of assays that work well with antibodies.               


Analytical applications:

The development of sensitive and high throughput assays for multiplexed protein detection remains an extremely important goal in drug discovery and clinical diagnosis. Numerous aptamers against a wide range of protein targets have already been selected and have affinities in the Pico molar to low Nano molar range. Aptamers have shown increasing utility as affinity reagents and can successfully compete with antibodies in a number of analytical application.27


 Aptamers are very attractive as replacement for antibodies in affinity analysis of proteins .Aptamers  have several advantages over antibodies.28They  are smaller ,more stable can be chemically synthesized and can be fluorescently labeled  without affecting their affinity 13,29,30,31


The use of aptamers as tools in analytical chemistry is  very promising and exciting field of research due to their capabilities to bind specifically to target molecule. SELEX procedure has allowed the discovery of aptamers against various targets such as ions, small organics, positively and negatively charged peptides and proteins, viruses and tissues.13 The in vitro selection conditions (buffer composition, temperature, etc.) can be manipulated to obtain binding properties desirable for specific assays as the specific binding pocket of the aptamers can be elucidated by chemical and enzymatic foot printing. It is also possible to change their sequence at critical, precise location in order to modulate the selectivity and kinetic parameters of binding.


The aptamers can be produced by chemical synthesis at relatively high degree of purity resulting in little or no batch to batch variation and reporter molecules can be attach to aptamers at precise location. Thus, they are produced through an in vitro process which does not require animals.32 Taken into account all these background features, various analytical aptamers based formats have been exploited including enzyme linked oligonucleotide assays, biosensors(aptasensors), aptazymes, flow cytometry33-36 . The applications of aptamers as specific legands in liquid chromatography, electro chromatography, and capillary electrophoresis have also received much attention during the last few years.


1) Aptamers as ligand for chromatographic and electro chromatographic applications:- 32

Although various ligands have been used in Affinity chromatography, the most popular format uses the high affinity and specificity of antibodies to create efficient immunoaffinity columns. However, there are some constraints that reduce the effectiveness of antibodies. The linkage of antibodies to column often results in couplings that are not uniform, leading to reduced binding capacity and can allow the leaching of the antibody from the column.



Fig. No. 3: Possible modification on an oligonucleotide strand to generate modified oligonucleotide libraries for the SELEX process.


Fig. No. 4:  Schematic representation of principle behind the Ligand beacon assay designed to detect target molecules in a competitive and homogeneous manner.



More, antibodies present relatively high size which limits the ligand density at the chromatographic surface. Finally the elution conditions can be harsh, requiring extremes of pH, detergents, organic solvents or chaotropic salts leading to denaturation of the antibody and possibly the target (e.g. protein) Expected advantages of aptamers relative to antibodies for affinity chromatography include smaller size enabling higher density stationary phases, novel approaches for elution and possibility to immobilize the the ligand to chromatographic surface at a precise location.  Moreover, a number of recent reports have shown the great interest in use of immobilize aptamers (DNA or RNA) as affinity ligands in chromatography or electro chromatography. To date they have been applied to the separation or purification of proteins and separation of small molecules including enantiomers.


2) Target specific chiral separation:

For chiral compounds, the efficient monitoring of the selection procedure has allowed in most of cases a very high specificity exemplified by capability of aptamers to bind enantio selectively a target.  Such chiral discrimination properties of Aptamers selected against a target enantiomers have been accounted to create a new class of target specific chiral stationary phases.


This approach has been extended to the chiral resolution of small molecules of biological interest. A necessary condition is that an efficient SELEX procedure for high affinity binding would result in the isolation of enantiospecific oligonucleotide. Thus, the target immobilization to the matrix must allow an adequate exposure of the enantiomer key functional groups to the oligonucleotide pool during the in vitro selection. 37-38


Fluorescence Labeled Aptamers:-

Fluorescence Labeled Aptamers are synthesized by a combination of pre and post synthetic modification methods. The Fluorescence Labeled Aptamers has binding affinities nearly equal to that of original Aptamers. It is sensor detected specific biomolecule and has an important application in chemical biology. These sensors can detect biologically active molecules and determined the concentration of these molecules.31A molecular sensor consist of a recognition moiety and a signaling moiety. One of the most useful recognition molecules is an aptamer 39 and fluorescence molecules are attractive as a signaling moiety. Therefore if the Aptamers are labeled with a fluorophore, the fluorescent labeled Aptamers are used for signaling Aptamers. Two kinds of signaling Aptamers have been reported. One is a single fluorophore labeled DNA. This type of probe exhibits changes in fluorescence upon binding to a target molecule and this change in fluorescence is based on the change in the micro -environment around a fluorophore. Other type of probe is a doubly labeled DNA bearing a fluorophore and fluorescence quenchers called molecular beacons.  Molecular becons are simple hairpin loop probes in which a fluorophore is attached to one terminus and a quencher is attached to the other. (See Fig No. 4) This mode of attachment brings the fluorophore closed to the quencher when the molecule beacons are folded into hairpins; the fluorescence is then quenched by the formation of a non fluorescent complex between the fluorophore and the quencher.40 The nucleic acid sequence in the loop of molecular beacons, is designed to be complementary to the target of interest. The loop of the molecular beacons interacts with its target sequence to form an intermolecular hybrid, during which the stem of beacon unfolds to move the fluorophore away from the quencher. The end result is the emission of fluorescence from the previously non fluorescent molecular beacon. Alternatively, Aptamers that change the fluorescence characteristics of a fluorophore attached either to the Aptamers themselves (for non-competitive assays) or to the analyte (for competitive assays) would be useful. In fact, Aptamers directed to bind flavins have been shown to quench the fluorescence of the target when it is bound by the Aptamer.41 Thus it is possible that targets that inherently fluoresce could be detected by Aptamers by measuring the changes in fluorescence.


Therapeutic Applications:

Aptamers in cancer treatment: Aptamers should be better suited for rapid tumor penetration and blood clearance due their relative small size (8-15 kDa). Changes in the expression of key protein of the cellular signaling pathways are at the forefront of molecular abnormalities found in cancer. An increasing number of proteins involved in cell growth, including growth factor receptor, intracellular mediators and transcription factor have been found to be altered through  multiple mechanism of oncogene activation, such as enhanced or ectopic expression, deletions, single point mutations and generation of chimeric proteins, the latter being the consequence of chromosomal translocations.


All these proteins represents primary target in the rational approach of the cancer mechanisms as major molecular determinants of cancer cells. Hence finding specific ligands capable to detect and measure their expression is a strategic objective for the diagnosis and therapy of cancer. Owing to high affinity and selectivity Aptamers are being identified as powerful antagonist of proteins which are associated with tumor.42 NeXstar  Pharmaceuticals isolated RNA Aptamers to the vascular endothelial growth factor (VEGF 165) isoform with 2’-o-methyl purine and 2’-F pyrimidines, in 1995 and 1998 respectively.43 VEGF Aptamers can lead not only to regression of tumor vessels, but also exhibit a remarkable stability in plasma in monkeys.44Macugen (Pegaptanilo sodium) targeted against the (VEGF 165) , was approved by the FDA in 2004 for the treatment of neovascular macular degeneration.  45, 46 and is in phase-I clinical trials.


Intramers and their therapeutic applications:

RNA Aptamers with 2-fluoro pyrimidine modifications, 2-o-methyl nucleotides and 3 end cap modification significantly enhanced the stability of Aptamers in vivo.47Aptamers themselves are very small are rapidly eliminated from the body by renal clearance. Further modifications, using cholesterol and polyethylene glycol as an anchor groups, have been shown to improve the bioavailability and the pharmacokinetics parameters of Aptamers.48 It is also becoming increasingly evident that Aptamers can not only be expressed inside cells, but also retain their functions and can alter the phenotype of a cell by modulating the biological functions of the targeted protein. Because of their intracellular mode of action the term “intramer " was coined for these type of nucleic acid molecule. Intramers can be easily modified and improved, for example, to specifically distinguish between proteins that are highly homologous.


Several groups have evolved Aptamers against proteins essential for HIV-1 replication, such as HIV-1 reverse transcriptase, integrase and reverse protein.49To develop highly specific drugs with minimal side effects it will become increasingly important to a particular dysfunction. Intramers can easily be tailored for their use in vivo by adding additional functions, such as enhanced stability and signals for cellular localization.


Aptamers to treat autoimmune diseases: 50-51

Sometimes human immune system fails to distinguish between self and foreign antigens and reacts by formation of auto antibodies. These give autoimmune diseases like Myasthenia gravis, insulin dependant diabetes mellitus, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and hemolytic anemia. Standard therapeutic approaches to autoimmune diseases usually involve symptomatic palliation with anti-inflammatory drugs and general immuno-suppression often causes toxic side effects. Several experiments are being carried out to investigate the therapeutic potential of RNA aptamers against auto antibodies. Their results suggest that RNA aptamers could be applied for antigen specific treatment for autoimmune diseases including Myasthenia gravis.



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Received on 20.03.2009       Modified on 20.05.2009

Accepted on 26.06.2009      © RJPT All right reserved

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 449-455