Application of locked nucleic acids to improve aptamer in vivo stability and targeting function

Abstract

Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure-activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degradation. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2'-O-methyl RNA (2'-OMe), 2'-Fluoro RNA (2'-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex. TTA1 derivatives with LNA and 2'-OMe modifications within the non-binding stem have subsequently been synthesized. Especially, the LNA-modified TTA1 derivative exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addition, higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties.

Figure 1

Structure of the TTA1 aptamer represented by 39 nt with a thymidine cap at the 3′ end and a MAG₂ chelate conjugated via an hexyl-aminolinker at the 5′ end. All pyrimidines are 2′-F nucleotides and all purines with the exception of the indicated guanosines are 2′-OMe nucleotides. The supposed stem regions I, II and III are plotted as ellipses exclusively in the (CH₂CH₂O)₆-loop.

Figure 2

Melting curves of TTA1 (dotted), TTA1.1 (straight) and TTA1.2 (interrupted) recorded at 260 nm between 10 and 90°C in a low-salt buffer (1 mM sodium phosphate, 0.1 mM EDTA). Curves were plotted with GraphPadPrism 3.02 using a Boltzman-sigmoidal regression fit.

Figure 3

Analysis of the stability of TTA1 and its derivatives in human blood plasma by PAGE (see Materials and Methods). The two spots of TTA1.2 and TTA1.4 are most likely due to alternative conformations.

Figure 4

Binding curves of TTA1 (circles), TTA1.1 (triangles), and TTA1.2 (squares) to human Tenascin-C competing against Tc-99m-TTA1 with concentrations between 10⁻¹² and 10⁻⁶ M. Curves were plotted with GraphPadPrism 3.02 using a one-side-binding regression fit of the means (n = 3, ±SD).

Figure 5

Blood kinetics (A) and tumor uptake (B) of TTA1 (circles), TTA1.1 (triangles), and TTA1.2 (squares) labelled with Tc-99m in U251-tumor-bearing nude mice. Values are expressed as % injected dose per gram (n = 3, ±SD).

Figure 6

Biodistribution of Tc-99m-labelled TTA1 (white), TTA1.1 (stripe), and TTA1.2 (traverse) in U251-tumor-bearing nude mice. Values are expressed as % injected dose (n = 3, ±SD) measured 1 h post injection.