LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1

Abstract

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.

Figure 1

Nucleic acid monomers and miniTAR–aptamer kissing complex. (A) Schematic representation of the sugar chemical structure adopted by a DNA, RNA and LNA monomer. (B) Loop–loop complex formed between the R06₁₆ aptamer (bold) and miniTAR RNA used in this study. MiniTAR is an imperfect hairpin corresponding to the top part of the retroviral TAR element. The consensus octamer (5′-GUCCCAGA-3′) obtained from in vitro selection is shown in italic. The closing G and A residues of the aptamer are underlined. The potential base pairs are indicated (vertical lines).

Figure 2

Sensorgrams of LNA/DNA R06 derivative–miniTAR complexes. A total of 500 nM concentration of LNA5 (thin line), LNA4 (bold line) or anti-loop LNA5 (dotted line) were injected on a miniTAR-functionalized sensorchip. Rate constants, k_on and k_off, for bimolecular complex formation were deduced from direct fitting of these plots according to equations 1 and 2 (Materials and Methods). Three independent experiments were carried out in R buffer (3 mM Mg²⁺) at 23°C.

Figure 3

Stability of oligonucleotides in bovine serum. Oligonucleotides (RNA, DNA, LNA5 and anti-loop LNA5) derived from the aptamer R06₁₆ were incubated in bovine serum at 37°C. Aliquots were taken at the time point indicated and analysed on a 20% denaturing 7 M urea/polyacrylamide gel. Bands corresponding to the oligonucleotides were revealed by the Stains All procedure.