8.1 Introduction
The functional nucleic acids that specifically bind and regulate the cellular protein were first discovered in 1989.196 Interestingly, Turek and Gold then established a standard generation procedure for identification of RNA ligand with high affinity for T4 DNA polymerase as well as various organic dyes.197 Such functional nucleic acid molecules were termed as “aptamer” by Ellington and Szostak.198 Aptamer molecules were derived from an in vitro SELEX (Systematic
Evolution of Ligands by EXponential enrichment) selection.
197, 198 In general, SELEX consists of several iterative steps. The first step of SELEX process is to construct a single-stranded nucleic acid library. The chemically synthesized oligonucleotide containing a centralized random sequence segment (typical about 30 ~ 40 nucleotides) flanked with two fixed terminal sequences was directly used as the starting material library for the first round of DNA-SELEX or subjected for the reverse transcription to generate the material for the first round of RNA-SELEX. Due to the restricted chemical synthesis scale, the diversity of random library was commonly limited to 1015 distinct molecules even though the theoretical value could be composed of 1024 molecules (440 = 1.2 x 1024). However, this kind of initial library was big enough to generate a high probability for obtaining a desired aptamer. Second, this randomsequence library was incubated with the target molecule and the different washing strategies were subsequently chosen for separating the unbound oligonucleotides from the target/oligonucleotide complex. For example, for the small organic molecules which were pre-immobilized on the solid supporting matrix, the buffer solution was enough to remove the unbound oligonucletides. For the protein targets, the fitter binding assay with nitrocellulose membrane or the gel mobility shift assay were usually selected to analyze the binding behavior between oligonucleotides and proteins. In addition, the affinity chromatographic column or the ELISA microtiter plates were also been utilized for the protein immobilization during the SELEX process. Subsequently, after dissociating the bound oligonucleotides from the target molecules via harsh or denaturing extraction, the standard ethanol precipitation was carried out to concentrate and purify the residual oligonucleotides. The next step is to amplify the resultant nucleic acids pool via the PCR–based reaction. Alternatively, the single-stranded DNA separation or the reverse transcription was performed to obtain the starting materials for the next round of SELEX selection. Theoretically, the next SELEX cycle utilized the newly generated nucleic acids library will enrich those oligonucleotides which have higher affinity for the same target molecule than that of previous SELEX round. The affinity of bound oligonucleotides exponentially increases for each round of SELEX cycles. After several repeating rounds of these stages, the nucleic acids with high affinity for the target molecules were isolated via general molecular cloning technology and followed with DNA sequencing analysis.
Interestingly, a derivative of SELEX technology, photo-SELEX, from which the substrate of either DNA polymerase or RNA polymerase was exchanged for the 5’-iodo or 5’-bromo dUTP or UTP in the PCR reaction has been included in the normal SELEX procedure.199-201 These evolved aptamer molecules allow the photo-crosslinking reaction occurred between target protein and the photo-aptamer
molecules after exposing them under long wavelength UV light. This kind of laser induced photo-crosslinking process has been mentioned recently to further facilitate the design of aptamer-assisting diagnostic array system.201, 202 In parallel, the random double-stranded DNA library has also been used in the SELEX procedure to identify the dsDNA consensus for the interest protein.203, 204 Moreover, in order to isolate nucleic acid aptamers with the extremely high specificity, the counter selection strategies or the negative selection strategies were broadly carried out during the SELEX procedure.205 On the other hand, the stringent selection conditions might also be applied to adjust the binding characteristic of the resultant nucleic acids.
Theoretically, the nucleic acid molecule with highest specificity and the highest affinity for the target molecule could be isolated and confirmed for its binding property via different biophysical studies. The “winner” of nucleic acid isolated from the in vitro section was named “aptamer”, which was originally derived from Latin word “aptus”, meaning “to fit”. Moreover, after completely sequencing of the full-length of aptamer, the essential region for the binding could be determined via design of the different truncate forms of aptamer. Usually, the nucleotide sequences that are not important for direct binding with target or for facilitating the correct structure folding should be eliminated from the full-length. The scheme for the selection of DNA aptamer to the specific target molecule is shown in Figure 8.1.
Interestingly, the specificity of existing aptamer could also be further changed for another target molecule via the second selection of SELEX for the cognate targets by using a biased library which was produced from the existing aptamer.206 For example, the specificity change from L-arginine to L-citrulline of definite L-arginine-aptamer molecule could be used to demonstrate the power of the in vitro selection for optimizing or modifying the specific aptamer on demand.206, 207
Figure 8.1 The schematic diagram of the scelection of DNA aptamer to target molecule, i.e. the general SELEX procedure.
As we know, for a given target molecule, both antibody and aptamer exhibiting comparable binding affinity as well as high specificity could be theoretically isolated.
However, the production of antibody was derived from the in vivo immunoreactions, whereas the aptamer was derived from the in vitro SELEX procedures. Moreover, aptamers could also be obtained via chemical synthesis. Thus, different reporter molecules such as biotin, radioisotope, fluorophores, could be easily modified on the skeleton of aptamer molecules. Aptamer molecules could also specifically targeted at the non-immunogenic toxic molecules and be applied for the non-physiological selection condition. Hence, the target range of antibody was narrower than that of the
Biotin Molecular cloning and Squencing
of the Resulant Aptamer
PCR Reaction for Next SELEX Round
aptamer molecule. Moreover, the batch to batch variation of definite antibody caused by the in vivo immunoreactions process was significantly bigger than that of relative distinct production of aptamer. Further, the thermal denaturation was also reversible for an aptamer molecule. Importantly, the specificity and discrimination for an aptamer could further be regulated easily during the aptamer screening procedure.
Thus, the application and stability of aptamer is broader than that of antibody molecules. The advantages of aptamer over antibody and other unique characteristics of both molecules are listed in Table 8.1.
Table 8.1 The Comparison of functional characteristics between aptamers and antibodies.208
Aptamers Antibodies
Affinity Low nM - pM Low nM - pM
Specificity High High
Production In vitro process In vivo process
Target range Wide: ions, small organic molecules, protein, whole cells, toxic molecules, etc
Narrow: only immunogenic compounds
Batch to batch variation Little Significant
Chemical modification Easy and straightforward Limited
Thermal denaturation Reversible Irreversible
Shelf-life Unlimited Limited
Up to date, various targets including organic dye209, amino acids210, biological cofactors211, antibiotics212, peptides213, proteins214, even complex mixtures such as whole virus particle215, 216, cell membrane217, live trypanosomes218, and whole mammalian endothelial cell219 have been selected to isolate their own aptamers from diverse library, further indicating that the aptamer molecules have the property of broad target range. The online aptamer database listing all of the selected aptamers was publicly available on the internet (http://aptamer.icmb.utexas.edu/). The high
to be used as the recognition elements in the diagnostic application. The first fluorescent-tag modified aptamer was utilized in the flow cytometry system in 1996.220 After then, different recognition elements involving the functional aptamer molecules were dramatically developed. These engineered aptamer molecules have been employed in the diverse array of diagnostic systems containing the quartz crystal microbalance221, surface plasmon resonance technique222, electrochemical sensor223-228, and different FETs fabricated with single-walled carbon nanotubes (SWNTs)229, 230. Among these, the electrochemical sensors coupled with functional aptamers, compared to that of antibody-form biosensors, have another obvious advantage during the regeneration process. The bound protein could be easily removed from the electrode surface without damaging the sensing aptamer molecule itself, whereas the harsh washing step would denature the bound antigen as well as the antibody in the antibody-form biosensors. Hence, the repeatable utilization and reversible stability of aptamer molecules provide the superiority over the diagnostic systems based on the antibody.
Conversely, many aptamer molecules not only display high specificity and high affinity for the particular target but also disrupt the respective biological activity of this target. Thus, the aptamer molecules are also been referred as one of the prime potential drug candidates. For the purpose of application of aptamers in pharmaceutical medicine, the stability of nucleic acids is the critical issue for prior consideration. The endonuclease-resistant aptamer was developed after changing the 2’-OH group of ribose to 2’-NH2 or 2’-F modifying sugars on the pyrimidines moiety.
This kind of chemical modification provided not only the stability of aptamer but also the compatibility for enzymatic reaction in the SELEX protocol.231 Moreover, the phosphorothiolate linkage modification232 or the chemically synthesized enantiomer aptamer based on the parent-SELEX against the enantiomer of target provided