Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers.

G-quadruplex (G4) is a non-canonical structure that is formed in G-rich sequences of nucleic acids. G4s play important roles in vivo, such as telomere maintenance, transcription, and DNA replication. There are three typical topologies of G4: parallel, anti-parallel, and hybrid. In general, metal cations, such as potassium and sodium, stabilize G4s through coordination in the G-quartet. While G4s have some functions in vivo, there are many reports of developed applications that use G4s. As various conformations of G4s could form from one sequence depending on varying conditions, many researchers have developed G4-based sensors. Furthermore, G4 is a great scaffold of aptamers since many aptamers folded into G4s have also been reported. However, there are some challenges about its practical use due to the difference between practical sample conditions and experimental ones. G4 conformations are dramatically altered by the surrounding conditions, such as metal cations, pH, and crowding. Many studies have been conducted to characterize G4 conformations under various conditions, not only to use G4s in practical applications but also to reveal its function in vivo. In this review, we summarize recent studies that have investigated the effects of surrounding conditions (e.g., metal cations, pH, and crowding) on G4 conformations and the application of G4s mainly in biosensor fields, and in others.

To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer.Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimerhas a K_d value which is 1/10 of that of the monomers from which it is composed. Also, thedesigned aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers againstvascular endothelial growth factor (VEGF₁₆₅), which is a homodimeric protein. As in thecase of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower K_d value than that of the monomer. This study demonstrated that the dimerization of aptamerseffectively improves the affinities of those aptamers for their targets.