Aptamers As Biomolecular Recognition Elements Biology Essay

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Aptamers are short single-stranded oligonucleic acids. These oligonucleic acids have tendency to fold in to distinct and diverse three dimensional shapes1. Based on their 3D structures aptamers bind to target (small molecules and proteins) with high affinity and specificity. Figure 1 shows a schematic representation of the functionality of aptamers.1 Aptamers are considered as synthetic antibodies. In the year 1990, Dr Andrew Ellington and Dr Szostak coined the term aptamers.2 Aptamers bind with strong affinities to target molecules with dissociation constants in the nanomolar to picomolar range.3 Further, Aptamers are classified as RNA and DNA aptamers. Both RNA and DNA aptamers frequently fold into complex 3D structures. A 15mer DNA aptamer was one of the earliest studied structurally against thrombin.4 RNA and DNA aptamers are well suited as biosensors and are a good tool for therapeutic, diagnostic and analytical applications.5

In 1990, two groups independently developed a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the synthesis of aptamers in vitro.2, 6 Selection, partition, elution and amplification are the basic steps of the SELEX process. Initially a pool of randomized oligonucleotides is allowed to bind with the target for proper selection. Target bound nucleotides are then partitioned from unbound nucleotides. These bound nucleotides are eluted and later amplified. After amplification, these oligonucleotides are sent for another round of SELEX. By multiple repeated cycles of SELEX, the initial random oligonucleotide pool is reduced to relatively few sequences showing highest affinity and specificity for the target. Figure 2 shows the basic steps of: the SELEX process.6

Figure 2. SELEX: Sytematic Evolution of Ligands by Exponential Enrichment.6

Advantages of Aptamer

Aptamers are an emerging class of molecules with several important advantages over antibodies. First, aptamers are efficient at binding not only to large molecules (proteins, cells) but also to small molecules (ATP, Cocaine), whereas antibodies are mostly efficient at binding to large molecules. Second, aptamers are selected in vitro under non-physiological conditions while antibodies are produced in vivo under physiological conditions. Third, once an aptamer is selected, it can be obtained in a large amount. Fourth, the size of aptamers is much smaller (~ 1-2nm, < 10kDa) than those of antibodies (~ 10nm & ~ 155kDa). Finally, aptamers are stable to heat, pH and organic solvents. Table 1 summarizes the advantages of aptamers over antibodies.7

Table 1. Advantages of aptamers over antibodies.7

 

Aptamers

Antibodies

Affinity

Low nM-pM

Low nM-pM

Specificity

High

High

Production

In vitro chemical process

In vivo biological system

Target range

Wide: ions, small molecules, proteins, whole cells, etc

Narrow: only immnunogenic compounds

Batch to batch variation

Little or no

Significant

Chemical modification

Easy and straightforward

Limited

Thermal degradation

Reversible

Irreversible

Shelf-life

Unlimited

Limited

Aptamer targets

Aptamers bind efficiently to large molecules (proteins), small molecules (ATP, Cocaine), organic dyes, amino acids, cells and viruses. Aptamers show a potential niche market in diagnostics and drug delivery when small molecules are the targets8, due to the following reasons: a) Aptamers are selected in vitro and can be used to select a wide range of targets including toxic and non-immunogenic molecules. b) Selected aptamers can be produced in large quantity via chemical synthesis, and c) The selection process is performed under non-physiological condition. My seminar will mainly focus on small molecule targets such as ATP9, 10 and cocaine.11

Aptamers for biosensing

A biosensor consists of several types of transducers that utilize aptamers as the biomolecular recognition unit for sensing12. These include electronic, electrochemical, mass, thermal and optical methods. The first use of an aptamer as a biosensor was reported in 1996.13 They detected the recognition interaction using fluorescent tagged aptamers. Since then various transducers using aptamers have been reported. Electrochemical sensors show several advantages over other sensors. My talk will concentrate on electrochemical sensors due to their relatively low cost, simplicity, sensitivity and ease of miniaturization. There are several strategies or approaches involved in designing an electrochemical aptasensor. The first is a sandwich based strategy. The first aptamer-based electrochemical sensor was fabricated as a sandwich-based structure by using an aptamer as the recognition ligand.14 This strategy offers the advantages of high sensitivity and simple operation for biosensor fabrication. The target should have two or more recognition elements including the aptamers. The other recognition element serves as a probing element to mark the target with electroactive molecules or nanoparticles. Dong et al15 used this strategy for the detection of ATP and cocaine. They developed a probe label-free electrochemical technique to improve the sensitivity of the aptasensor. The second strategy is a conformational or structure switch strategy. This strategy measures a change in the electrochemical current caused by a reduction in the electron transfer distance of the electroactive probe from the aptamer sequence end to the electrode surface, or by desorption of electroactive intercalators from the aptamer bases. Li et al16 developed an aptasensor based on a conformational switch for the detection of adenosine. This method is cost effective compared to the more commonly used HPLC method. A third strategy is the target molecules displacement strategy. Displacement is a newer method using aptamers for electrochemically sensing biomolecules. There are several displacement schemes such as using an ion-selective field effect transistor (ISFET) and a triggered displacement strategy. In 2006, Joseph Wang’s group17 developed a new strategy using quantum dots. The aptasensor was based upon the displacement of the quantum dot tags on the probing proteins. Based on this strategy, Yuan et al18 recently developed a sensor for the simultaneous electrochemical detection of multiple small molecules. Nanomolar detection limits of small molecules could be achieved in complex serum samples. Recently, a new method for the simultaneous detection of small molecules based on a microfluidic platform has been developed.19 The microfluidic approach shows the advantage of using only one kind of label-free electrochemical probe. The combination of microfluidics and aptamers conveniently demonstrated the concept of on-chip analysis. There are several other strategies used for electrochemical sensing but those listed here are most commonly used.

Future directions

In 2004, a therapeutic aptamer named Macugen received approval from the USFDA for the treatment of all types of neovascular age-related macular degeneration (AMD).20, 21 Other therapeutic aptamers in clinical development to treat are: 1) lung cancer, melanoma, cutaneous T-cell lymphoma, 2) hepatitis C and HIV, 3) asthma and allergy, and 4) blood coagulation during surgery (short half-life anticoagulants/ antithrombotics). The studies on aptamer research is relatively considered to be in its developmental stage, but it is progressing at a fast pace. The value of aptamers will be in applications where the performance of antibodies is inadequate. This benefit will be of great value in near future with more research and development in therapeutic and diagnostic field.

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