Diversity of Self-Assembled RNA Complexes: From Nanoarchitecture to Nanomachines

Abstract

New tool development for various nucleic acid applications is an essential task in RNA nanotechnology. Here, we determined the ability of self-limited complex formation by a pair of oligoribonucleotides carrying two pairwise complementary blocks connected by a linker of different lengths in each chain. The complexes were analyzed using UV melting, gel shift assay analysis, and molecular dynamics (MD) simulations. We have demonstrated the spontaneous formation of various self-limited and concatemer complexes. The linear concatemer complex is formed by a pair of oligomers without a linker in at least one of them. Longer linkers resulted in the formation of circular complexes. The self-limited complexes formation was confirmed using the toehold strand displacement. The MD simulations indicate the reliability of the complexes' structure and demonstrate their dynamics, which increase with the rise of complex size. The linearization of 2D circular complexes into 1D structures and a reverse cyclization process were demonstrated using a toehold-mediated approach. The approach proposed here for the construction and directed modification of the molecularity and shape of complexes will be a valuable tool in RNA nanotechnology, especially for the rational design of therapeutic nucleic acids with high target specificity and the programmable response of the immune system of organisms.

Keywords: RNA; circular RNA; concatemer; molecular dynamics of nucleic acids; rational design; self-assembly; self-limited complex; supramolecular complexes of nucleic acids.

Figure 1

Schematic representation of the complexes’ formation. (a) Concatemer complex; (b) self-limited complexes with different duplex numbers; (c) formation of linear trimolecular complex from self-limited complex by adding an opener (O); (d) formation of self-limited complex from linear with the opener by adding a closer (C). The pink block is complementary to the violet block, the light-blue block is complementary to the dark-blue block. M is the name of an oligonucleotide containing pink and light-blue blocks, and N is the name of an oligonucleotide containing violet and dark-blue blocks. M-Ui and N-Uj are the names of sequences M and N with a linker length of i and j uridines consequently. Formation of complementary base pairs schematically marked by thin red lines. Base staking interaction in nicks of concatemer is highlighted by orange.

Figure 2

The gel shift assay of oligonucleotides’ complexes M-Ui/N-U3 with various lengths of linkers (i = 0, 1, 2, 3, 5, 7, 10, or 15). Lanes 1—M/N-U3; 2—M-U1/N-U3; 3—M-U2/N-U3; 4—M-U3/N-U3; 5—M-U5/N-U3, 6—M-U7/N-U3; 7—M-U10/N-U3; 8—M-U15/N-U3. The types of complexes are labeled below the lanes and shown on the right. A dsDNA ladder of 50–1000 bp is shown on the left. The color designations of oligonucleotides and complexes are identical to those shown in Figure 1.

Figure 3

Determination of self-limited complex molecularity by adding the opener (O). Gel shift assays of M-U5/N-U3 complexes complex in the presence of RNA-opener O. Lanes: 1, M-U5/N-U3 (1:1); 2, M-U5/N-U3/O (1:1:0.1); 3, M-U5/N-U3/O (1:1:0.25); 4, M-U5/N-U3/O (1:1:0.5); 5, M-U5/N-U3/O (1:1:0.75); 6, M-U5/N-U3/O (1:1:1); 7, M-U5/N-U3/O (1:1:2); 8, M-U5/N-U3/O (1:1:5); 9, M-U5/N-U3/O (1:1:10). In the brackets the ratio of components concentration in the sample noted. Value 1 corresponded to 10 μM. The types of complexes are labeled and shown on the right. A dsDNA ladder of 50–1000 bp is shown on the left. The color designations of oligonucleotides and complexes are identical to those shown in Figure 1.

Figure 4

Heat map showing the types of complexes formed by a pair of oligonucleotides M-Ui and N-Uj with various linkers length: concatemer (red); bimolecular complex (salmon); tetramolecular (light green); high-molecular self-limited complex (green). If a cell has two colors, it means that the molecularity for it is not unambiguous. k is the number of duplexes in the complex; i and j is the linker length. The upper location red triangle in the cells indicates the predominant formation of concatemer, lower location—domination of self-limited complex(es). The color designations of oligonucleotides and complexes are identical to those shown in Figure 1.

Figure 5

Molecular structures of the studied complexes most represented in MD trajectories: (a) M-U3/N-U2, (b) M-U5/N-U2, (c) (M-U3/N-U2)₂, (d) (M-U3/N-U3)₂, (e) (M-U3/N-U3)₆. Linkers are shown as green, oligonucleotides of the M series are shown with blue backbone, N series with red backbone. The structures obtained by hierarchical cluster analysis.

Figure 6

Verification of the reversible changes from cyclic complexes to linear and vice versa. (a) Gel shift assays of M-U5/N-U3 complex in the presence of elongated opener OL and elongated closer CL. Lanes: 1, M-U5/N-U3 (1:1); 2, M-U5/N-U3/OL (1:1:1); 3, M-U5/N-U3/OL/CL (1:1:1:1); 4, M-U5/N-U3/OL/CL (1:1:1:2). (b) Gel shift assays of M-U5/N-U1 complex in the presence of OL and CL. Lanes: 1, M-U5/N-U1 (1:1); 2, M-U5/N-U1/OL (1:1:1); 3, M-U5/N-U1/OL (1:1:2); 4, M-U5/N-U1/OL/CL (1:1:2:1); 5, M-U5/N-U1/OL/CL (1:1:2:2); 6, M-U5/N-U1/OL/CL (1:1:2:3); 7, M-U5/N-U1/OL/CL (1:1:2:5). In the brackets the ratio of components concentration is presented. Value 1 corresponded to 10 μM. The types of complexes are labeled on the right and illustrated in the middle. A dsDNA ladder of 50–1000 bp is shown on the left. The color designations of oligonucleotides and complexes are identical to those shown in Figure 1.