Triggering nucleic acid nanostructure assembly by conditional kissing interactions

Abstract

Nucleic acids are biomolecules of amazing versatility. Beyond their function for information storage they can be used for building nano-objects. We took advantage of loop-loop or kissing interactions between hairpin building blocks displaying complementary loops for driving the assembly of nucleic acid nano-architectures. It is of interest to make the interaction between elementary units dependent on an external trigger, thus allowing the control of the scaffold formation. To this end we exploited the binding properties of structure-switching aptamers (aptaswitch). Aptaswitches are stem-loop structured oligonucleotides that engage a kissing complex with an RNA hairpin in response to ligand-induced aptaswitch folding. We demonstrated the potential of this approach by conditionally assembling oligonucleotide nanorods in response to the addition of adenosine.

Figure 1.

Principle of conditional nanorod formation. The double-aptaswitch (DS) is in equilibrium between two structures (top line) upon the addition of adenosine (ado). The apical loops (blue) of the ado–bound structure engages kissing interaction with the loops (red) of the double-aptakiss (dk) leading to the formation of the nanorod. In the absence of adenosine no kissing takes place.

Figure 2.

Oligonucleotide toolbox. (A) aptakiss k; (B) double aptakiss dk; (C) control aptakiss k_m, which doesn’t bind to L; (D) hairpin L with a complementary loop to k; (E) adenoswitch S' (F) mutated double adenoswitch DS_mut; (G) double adenoswitch DS. Color code. Cyan blue: adenosine recognition internal loop; blue: kissing motif in S, L or DS loop; red: kissing motif in k or dk loop; green: point mutation, preventing kissing complex formation.

Figure 3.

Evaluation of the adenosine-dependent binding properties of the double aptaswitch DS to the aptakiss k_TR by fluorescence anisotropy. (A) k_TR: Texas Red-conjugated aptakiss (10 nM); ado: adenosine (1 mM); ino: inosine (1 mM); k_mTR: Texas Red^®-conjugated aptakiss variant (10 nM); DS: double adenoswitch (100 nM). (B) Binding curve of DS to k, as a function of adenosine concentration. Concentrations of k and DS as in A. See supporting information for methods and sequences (Supplementary Table S1).

Figure 4.

Non-denaturing PAGE shift assay of adenosine-dependent kissing complexes (silver nitrate staining). DS and dk (2 μM each) were run alone in lanes 1 and 2, respectively, and together (lanes 3) (A). In the absence of adenosine (ado), or (B). in the presence of inosine (ino, 8 mM) in the gel, or (C). in the presence of adenosine (8 mM) in the gel.

Figure 5.

Adenosine-dependent formation of DS–dk nanorods monitored by AFM. Complexes of DS and dk (1.25 μM each) were analyzed in the absence (left) or in the presence of 4 mM of inosine (centre, as a control) or 4 mM adenosine (right). Scale bar: 100 nm.