Nucleic Acid aptamers: new methods for selection, stabilization, and application in biomedical science

Abstract

The adoption of oligonucleotide aptamer is well on the rise, serving an ever increasing demand for versatility in biomedical field. Through the SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamer that can bind to specific target with high affinity and specificity can be obtained. Aptamers are single-stranded nucleic acid molecules that can fold into complex threedimensional structures, forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target. Recently, aptamers have attracted much attention because they not only have all of the advantages of antibodies, but also have unique merits such as thermal stability, ease of synthesis, reversibility, and little immunogenicity. The advent of novel technologies is revolutionizing aptamer applications. Aptamers can be easily modified by various chemical reactions to introduce functional groups and/or nucleotide extensions. They can also be conjugated to therapeutic molecules such as drugs, drug containing carriers, toxins, or photosensitizers. Here, we discuss new SELEX strategies and stabilization methods as well as applications in drug delivery and molecular imaging.

Keywords: Aptamer; Drug delivery system; Imaging; Modification; SELEX; Stabilization.

Fig. 1.. Overview of SELEX scheme. Aptamers can be obtained through an iterative selection process known as SELEX (systematic evolution of ligands by exponential enrichment) by using single-stranded DNA or RNA. An initial pool of 10¹⁴-10¹⁵ random oligonucleotide (ONT) strands are subjected to binding with the target. Unbound ONTs are discarded and RT-PCR or PCR is performed to amplify the targetbound ONTs. This selection process is repeated 6-15 times using amplified ONTs as a new pool. This way, aptamers having high specificity and affinity are screened. Diverse molecules can be the target of the SELEX, including metal ion, protein, organic compound and cell. Toggle-SELEX performs SELEX with two different target molecules to obtain bispecific aptamers.

Fig. 2.. Method for obtaining Spiegelmer. SELEX is performed with mirror-imaged target and D-form RNA. Based on the selected D-form RNA sequence, L-form RNA aptamer can be synthesized. Now L-RNA, the chiral form of the acquired D-form RNA, can bind to natural target. This L-form RNA is nuclease-resistant and suitable for in vivo application. The world ‘Spiegelmer’ is derived from German ‘Spiegel’ meaning ‘mirror’.

Fig. 3.. Structure of chemically modified nucleotides (Modified form Kasahara and Kuwahara, 2012). The simplest ribose 2’ modification is widely used to increase aptamer stability in vivo. Phosphate and base modification are also used for this purpose. Substitution of F, OCH₃, SH or CH₂OH for 2’-OH (H) is widely used. BNA/LNA was designed to structurally protect 2’ site. In addition, thiol (S) or borane (BH₃) group is introduced to α phosphate to strengthen oligonucleotide backbone. Functional groups can also be introduced into the base.

Fig. 4.. Applications of aptamer. Aptamer can be used as therapeutics, targeted drug delivery system (DDS) and imaging. These three parts can be put together using aptamer and conjugation in multifunctional probe.