No abstract

Aptamers are short single-stranded DNA or RNA oligonucleotides that can selectively bind to small molecular ligands or protein targets with high affinity and specificity, by acquiring unique three-dimensional structures. Aptamers have the advantage of being highly specific, relatively small in size, non-immunogenic and can be easily stabilized by chemical modifications, thus allowing expansion of their diagnostic and therapeutic potential. Since the invention of aptamers in the early 1990s, great efforts have been made to make them clinically relevant for diseases like macular degeneration, cancer, thrombosis and inflammatory diseases. Furthermore, owing to the aforementioned advantages and unique adaptability of aptamers to point-of-care platforms, aptamer technology has created a stable niche in the field of in vitro diagnostics by enhancing the speed and accuracy of diagnoses. The aim of this review is to give an overview on aptamers, highlight the inherent therapeutic and diagnostic opportunities and challenges associated with them and present various aptamers that have reached therapeutic clinical trials, diagnostic markets or that have immediate translational potential for therapeutics and diagnostics applications.

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.

Vascular endothelial growth factor (VEGF165) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF165 protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF121, an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF165 protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.