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Bioluminescent Binding Microassay Using Aptamers as Biospecific Elements

High specificity is an important requirement for an analytical system aimed at identifying a specific molecular target. Traditionally, antibodies, haptens and some other molecules are used for this purpose. Recently, aptamers were proposed as biospecific elements. Aptamers are short singlestranded oligonucleotides with a unique spatial structure that enables them to recognize target molecules and bind to them. Aptamers are obtained from synthetic random DNA(RNA)-libraries, a pool of oligonucleotides of the same length with different base sequences (1014-1015 variants), by selecting the oligonucleotides that are capable of specific binding to a given target. Aptamers are stable molecules with high affinity and specificity; they can be developed for any target, including toxic and nonimmunogenic ones; and they can be easily synthesized chemically. Due to these useful qualities, aptamers are often considered to be an alternative to antibodies. This paper describes the use of aptamer sensors and a highly sensitive bioluminescent reporter, the Ca2+-regulated photoprotein obelin, for the detection of diagnostically important targets in the blood of patients. Additionally, obelin was successfully applied as a reporter in the process of obtaining aptamers. A proposed bioluminescent solid-phase assay enables the enrichment of the oligonucleotide library with target-specific oligonucleotides to be monitored rapidly, the affinity of individual aptamers and their shortened variants to be evaluated and the relative position of the aptamers on the target molecule to be determined. The results of the studies reviewed in this paper open promising avenues for developing analytical systems that include highly specific aptamer sensing, as well as highly sensitive detection based on bioluminescent reporter proteins.


Introduction
The process of binding analysis includes recognition of the target with the formation of a specific complex, the appearance of which is detected by the various reporters (Fig. 1).
Some examples of reporters are radioisotopes, fluorophore molecules, and enzymes that generate visually detectable (or measurable) products (e.g., color and light).
In this study, we discuss a bioluminescent reporter, that is, an enzyme generating a quantum bioluminescence is triggered by Ca 2+ , which is why it is referred to as a 'Ca 2+ -regulated' protein.
Obelin has been successfully used as a reporter in bioluminescent binding assays for determining different diagnostically important targets, including hormones, oncomarkers, infection agents, and single nucleic polymorphisms (Frank, 2010). The assay provides highly sensitive and repeatable target detection with fast and simple signal reading, supporting that obelin is a reporter of choice.
To provide specificity to the binding assay, the label must contain a recognition element able to bind to the target with high affinity (Fig. 1).
Usually, this element is an antibody or a hapten binding protein (e.g., in the case of streptavidinbiotin interaction). Relatively new biospecific molecules are aptamers, single-stranded DNA or RNA oligonucleotides (20-80 bases long) that fold into certain structures, exhibiting high specificity when binding to the target of interest.
In the assay, aptamers function in a similar way to antibodies but successfully compete with them because aptamers are easily synthesized chemically (no animals needed) and can be selected for almost any target, including those that have low molecular weight and are toxic or nonimmunogenic. Due to these advantages, aptamers are becoming increasingly popular as biospecific elements in a number of biomedical analytical systems (Zhou et al., 2014). There are many methods (e.g., ultrafiltration, gel and capillary electrophoresis, HPLC, circular dichroism, and surface plasmon resonance) used to develop and characterize novel aptamers, which is usually a complicated and timeconsuming process. In this study, we consider a novel, rapid and simple method for aptamer development based on the obelin bioluminescent signal. Additionally, the paper describes several Recently, a group of 10 DNA aptamers specific to  Pathogenic autoantibodies to myelin basic protein (anti-MBP) were first reported in 1980 (Panitch et al., 1980). It was ascertained that anti-MBP to anti-MBP autoantibodies have been recently obtained Vorobjeva et al., 2014). To provide RNA resistance to ribonuclease cleavage, all pyrimidines in their sequences were replaced with 2'-fluoro analogues.
Based on these aptamers and obelin, a bioluminescent solid-phase sandwich assay for the pathogenic anti-MBP antibodies was developed (Fig. 5). One aptamer (Apt 12-2b, Fig. 5a) was adsorbed on the microwell surface and "worked" on the target capturing, whereas the other aptamer (Apt 2-9c) preconjugated with obelin was applied as a specific bioluminescent probe to reveal the complex.
The assay was first tested on model samples of hIgG at a constant concentration (1.5 mg/mL) spiked with anti-MBP autoantibodies (Fig. 5b).

Modern analytical technologies based on
aptamers, that is, biospecific molecules that can be obtained by chemical synthesis, easily modified and stored for a long time, have opened considerable avenues for researchers to create methods for identifying novel diagnostically important targets. Using bioluminescent reporters ensures a high-sensitivity analysis.
Taken together, these factors can promote early diagnosis of the disease, which significantly affects treatment success. The results of the preliminary experiments described in this report demonstrate the potential of developing new analytical approaches involving aptamer sensors and bioluminescent reporters.