Chemical modification improves the stability of the DNA aptamer GBI-10 and its affinity towards tenascin-C

Abstract

Aptamers are useful tools in molecular imaging due to their numerous attractive properties, such as excellent affinity and selectivity to diverse types of target molecules and biocompatibility. We carried out structure-activity relationship studies with the tenascin-C (TN-C) binding aptamer GBI-10, which is a promising candidate in tumor imaging. To increase the tumor targeting ability and nuclease resistance under physiological conditions, systematic modifications of GBI-10 with single and multiple 2'-deoxyinosine (2'-dI) or d-/l-isonucleoside (d-/l-isoNA) were performed. Results indicated that sector 3 of the proposed secondary structure is the most important region for specific binding with TN-C. By correlating the affinity of eighty-four GBI-10 derivatives with their predicted secondary structure by Zuker Mfold, we first validated the preferred secondary structure at 37 °C. We found that d-/l-isoNA modified GBI-10 derivatives exhibited improved affinity to the target as well as plasma stability. Affinity measurement and confocal imaging analysis highlighted one potent compound: 4A/26T/32T, which possessed a significantly increased targeting ability to tumor cells. These results revealed the types of modified nucleotides, and the position and number of substituents in GBI-10 that were critical to the TN-C binding ability. Stabilized TN-C-binding DNA aptamers were prepared and they could be further developed for tumor imaging. Our strategy to introduce 2'-dI and d-/l-isoNA modifications after the selection process is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding ability.

The Bioactivity of D-/L-Isonucleoside- and 2′-Deoxyinosine-Incorporated Aptamer AS1411s Including DNA Replication/MicroRNA Expression

“…Based on our previous experiences, selectively combinations of activity-enhanced sites are more likely to further improve the performance of the aptamer. 22 , 23 , 24 Additionally, 2′-dI offers a huge advantage for diagnostic utility, and when 2′-dI is incorporated at position 24, it can enhance the bioactivity of AS1411. To further optimize AS1411, we incorporate isoT at the 6 th and 12 th positions simultaneously (FCL-I).…”

“…Based on experimental results, D-/L-isoT increased the aptamer activities in a stronger way. We have used D-/L-isoNA to modify more than 5 different aptamers, including TBA, 21 AS1411, 25 GBI-10, 22 , 23 , 24 BC-15, and DT-2 (unpublished data). Some of these are G-quadruplexes, and some are not.…”

“… 17 Previous work has shown that D-/L-isoNA incorporated at the proper sites in oligos can increase the activity of antisense nucleotides, DNAzyme, small interfering RNAs (siRNAs), and aptamers. 18 , 19 , 20 , 21 , 22 , 23 , 24 Furthermore, as a naturally occurring base, 2′-deoxyinosine (2′-dI) ( Figure 1 ) offers a huge advantage in non-immunogenic clinical application. 25 With this strategy, the local spatial conformation, especially around the incorporation site, could be properly optimized, resulting in enhanced bioactivity.…”

Aptamers as Diagnostic Tools in Cancer

“…However, according to Rozemblum et al, backbone modifications can reduce the ability of polymerase to amplify the DNA in the PCR amplification step [ 53 ]. Conversely, the post-SELEX strategies introduce modifications to aptamers after the selection, during the solid-phase chemical synthesis of individual aptamers [ 54 ]. To solve the problem of renal filtration, aptamers are generally linked to polyethylene glycol (PEG), cholesterol, or proteins.…”

Applications of Aptamers in Cancer Therapy

“…The SMART probe had a better affinity and specificity to several tumors like glioma, prostate cancer, cervical cancer, and lung cancer cells [77]. The next generation of TN-C aptamer was GBI-10 and shown that it can bind several TN-C peptides in vitro [75,78]. The most important part of the fact that GBI-10 can target several binding sites of TN-C is the ability to address multiple splice variants at the same time.…”