Dependence of aptamer activity on opposed terminal extensions: improvement of light-regulation efficiency

Abstract

Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable ‘caged’ intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5′- to 3′-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation.

Optical Thermophoresis for Quantifying the Buffer Dependence of Aptamer Binding

“…However, Buff et al [25] also reported slightly reduced binding affinity resulting from 5'-extensions of the thrombin-aptamer. In SSC buffer the dissociation constant increases to K D = 190 AE 20 nm and in 50 % (10 %) human serum the binding was best fitted with the Hill equation, yielding EC 50 = 720 AE 100 nm (670 AE 80 nm) and a cooperativity of n = 2.…”

Polyvalent nucleic acid aptamers and modulation of their activity: a focus on the thrombin binding aptamer

“…In various strategies TBA 15 maintained its natural backbone and was only modified in the sequence: 1) by substituting several bases in the loops and/or in the G-quartets (Smirnov & Shafer, 2000; Saccà et (Uehara et al, 2008;Buff et al, 2010). Some researchers have exploited non-natural modifications of the nucleobases (Krawczyk et al, 1995;He et al, 1998a;Marathias et al, 1999;López de la Osa et al, 2006;Mendelboum Raviv et al, 2008;Nallagatla et al, 2009;Goji & Matsui, 2011), which included: 1) guanines modified with hydrophobic substituents in the N 2 and C 8 positions; 2) 6-thio-, 3) 8-amino-, 4) iso-, and 5) 8-bromo-guanine modifications; and 6) thymine with 4-thio-uracil substitutions.…”

“…Another strategy for the modulation of the anti-thrombin TBA activity was developed by Mayer's research group and consists in the insertion of photolabile units at defined positions of the aptamer sequence (caged aptamer) in order to exogenously regulate its inhibitory function using light, either by sterically blocking the interaction site with thrombin (Heckel & Mayer, 2005), or by inducing the formation (turn-ON) or depletion (turn-OFF) of the active conformation (Heckel et al, 2006;Mayer et al, 2009;Buff et al, 2010). In case of the steric blockage of the TBA binding site, a caged thymidine inserted in a key location (T4) is sufficient to completely mask the aptamer functions, i.e.…”

“…On the contrary, modified TBA 15 aptamers containing caged 5′ or 3′ sequence extension, upon UV-A irradiation, lost their anticoagulant activity (irreversible switch-OFF) as a consequence of the destruction of the G-quadruplex structure due to the release of the photolabile group: after that, the formation of a more thermally-favored intramolecular duplex structure (inactive form) was promoted (Fig. 11B) (Heckel et al, 2006;Buff et al, 2010). It is worth to underline that a caged aptamer with the 3′-extension presented, in its ON state, higher activity than the unmodified TBA 15 , and, most remarkably, also than a new generation aptamer, NU172 (Nuvelo Inc.), that is currently in Phase 2 clinical trials as an anticoagulant.…”

Translating Nucleic Acid Aptamers to Antithrombotic Drugs in Cardiovascular Medicine

“…gov, NCT00808964) but not yet recruiting patients. Furthermore, the design of more effective and efficient strategies to inhibit thrombin using aptamers with affinities for multiple thrombin binding sites has proceeded [38]. One approach has been to link an aptamer (HD1) with affinity for exosite I with a separate exosite 2 specific aptamer (HD22).…”