Affinity Improvement of a VEGF Aptamer by in Silico Maturation for a Sensitive VEGF-Detection System

Abstract

Systematic evolution of ligands by exponential enrichment (SELEX) is an efficient method to identify aptamers; however, it sometimes fails to identify aptamers that bind to their target with high affinity. Thus, post-SELEX optimization of aptamers is required to improve aptamer binding affinity. We developed in silico maturation based on a genetic algorithm (1) as an efficient mutagenesis method to improve aptamer binding affinity. In silico maturation was performed to improve a VEGF-binding DNA aptamer (VEap121). The VEap121 aptamer is considered to fold into a G-quadruplex structure and this structure may be important for VEGF recognition. Using in silico maturation, VEap121 was mutated with the exception of the guanine tracts that are considered to form the G-quartet. As a result, four aptamers were obtained that showed higher affinity compared with VEap121. The dissociation constant (K(d)) of the most improved aptamer (3R02) was 300 pM. The affinity of 3R02 was 16-fold higher than that of VEap121. Moreover, a bivalent aptamer was constructed by connecting two identical 3R02s through a 10-mer thymine linker for further improvement of affinity. The bivalent aptamer (3R02 Bivalent) bound to VEGF with a K(d) value of 30 pM. Finally, by constructing a VEGF-detection system using a VEGF antibody as the capture molecule and monovalent 3R02 as the detection molecule, a more sensitive assay was developed compared with the system using VEap121. These results indicate that in silico maturation could be an efficient method to improve aptamer affinity for construction of sensitive detection systems.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

“…Affinity for ligand is also important because intracellular ligand concentrations are expected to reach levels much lower than outside the cell. Tandem aptamers have been reported to have higher affinities than single aptamers for their protein ligands [23; 24]. However, 2xSPN2A showed no increase in affinity for DFHBI or PFP-DFHBI either in Buffer S (Fig.…”

Ultrasensitive SERS detection of VEGF based on a self-assembled Ag ornamented–AU pyramid superstructure

“…V 5 : 5′-SH-TTTTTTTGTGGGGGTGGACTGGGTGGGTACC-3′ (italics represent the VEGF 165 aptamer) (Nonaka et al, 2013).…”

“…It is well-known that the systematic evolution of ligands by exponential enrichment (SE-LEX), an efficient method for identifying aptamers, sometimes fails to identify the best aptamer to bind to the target (Nonaka et al, 2013). Fortunately, post-SELEX optimization of aptamers is now available to improve their binding affinity (Nonaka et al, 2013). The post optimization aptamers possess more advantages than antibodies such as chemical synthesis, easy modification, target versatility, high stability and resistance to degradation and denaturation.…”

“…In this study, a specific aptamer (Nonaka et al, 2013) for VEGF and its partial complementary sequence was used to assemble Ag ornamented-Au pyramid nanostructures. As shown in Fig.…”

An Update on Aptamer-Based Multiplex System Approaches for the Detection of Common Foodborne Pathogens

“…For target applicability, expression cassette (Martell et al 2002), TECS-SELEX (Ohuchi et al 2006), whole-bacteria SELEX (Torres-Chavolla and Alocilja 2009), mirror-image SELEX (Klussmann et al 1996), blended SELEX (Kulbachinskiy 2007), toggle SELEX target-switching (Hamula et al 2006), whole-cell SELEX (Park et al 2014b), complex target SELEX (Chen 2007) are more promising. Current emerged techniques such as HTS or automated SELEX (Huenniger et al 2014;Lu et al 2014), Non-or NECEEM-SELEX (Ashley et al 2012;Yun et al 2014), CE-SELEX (Kasahara et al 2013), FluMag-SELEX (Stoltenburg et al 2005a), microfluidic SELEX (Lin et al 2014), and in silico SELEX (Nonaka et al 2013;Savory et al 2013) have improved efficiency of designed aptamers in both binding affinity and high throughput in tertiary structure prediction, thermodynamic, and aptamer-target model (Das et al 2010). Despite all these enumerated SELEX techniques, some whole-cell-selected aptamer candidates would have high affinity but poor specificity (Hamula et al 2008).…”