Info

General Approach

The methodological approach [35, 104, 175] is described in Fig. 10.11. The rational design of artificial 3-D RNA architectures [35, 103, 104, 176] is based on an inverse folding process. Structural fragments corresponding to 3° structure motifs are ''cut and pasted'' from known X-ray or NMR structures (step 1) and inter-

Fig. 10.11 The RNA architectonics methodology. The process of engineering artificial tectoRNA architectures is a multistep procedure. First, RNA fragments extracted from known crystallographic or NMR data (step 1) are interactively reassembled into artificial RNA molecules by computer 3-D modeling (steps 2 and 3). These 3° models are then used as scaffolds to define consensus 2° diagrams (step 4) that are used as blueprints for designing

Fig. 10.11 The RNA architectonics methodology. The process of engineering artificial tectoRNA architectures is a multistep procedure. First, RNA fragments extracted from known crystallographic or NMR data (step 1) are interactively reassembled into artificial RNA molecules by computer 3-D modeling (steps 2 and 3). These 3° models are then used as scaffolds to define consensus 2° diagrams (step 4) that are used as blueprints for designing

RNA sequences (step 5) that are optimized by energy minimization [14] to maximize their thermodynamic stability and minimize the occurrence of alternative 2° structure folds [15]. The RNA sequences are then synthesized by chemical or enzymatic methods and characterized for their expected folding and self-assembly properties (step 6). TectoRNAs rational design can be optimized at the sequence or 3-D model levels (step 7).

actively reassembled into novel tectoRNA architectures by computer geometrical modeling with graphic user interfaces (step 2). During this mosaic modeling process [30], 3° interacting motifs can be positioned and oriented precisely by adjusting the lengths of their linking helical elements and the stacking of the helices at multi-helix junctions, thus allowing one to control the supramolecular assembly of RNA units. It is predicted that tectoRNAs will assemble into supramolecular architectures based on the conformation and geometry of their constitutive structural elements (step 3). These 3° models are then used as scaffolds to define consensus 2° diagrams, specifying invariant nucleotide positions to retain 3° structure constraints and positions involved in base pairing (step 4). TectoRNA sequences able to fold into these 2° blueprints are optimized by energy minimization [14] to maximize their thermodynamic stability and minimize the occurrence of alternative 2° structure folds [15] (step 5). The RNA sequences are then synthesized by chemical or enzymatic methods [35, 103] and their expected folding and self-assembly properties characterized by biochemical and biophysical methods like polyacrylamide gel electrophoresis (PAGE), temperature gradient gel electrophoresis (TGGE) and visualization techniques, such as AFM [35, 159, 175] or transmission electron microscopy (TEM) [104, 155] (step 6). The experimental data are then compared with the theoretical models and used to optimize the tectoRNA rational design at the sequence or 3° model level (step 7).

The effect and contribution of specific 3° structure motifs to the overall geometry and stability of the resulting supramolecular architecture can be assessed by introducing sequence mutations at key 3° nucleotide positions within tectoRNA molecules [35, 103, 176-179]. Mutated tectoRNA assemblies are used as negative control for comparison with non-mutated ones. Thus, this approach can also be a powerful way of unraveling the structural properties of 3° and 4° structure motifs for which few experimental data are available.

Although still a new field of investigation, RNA architectonics has already generated a great variety of tectoRNA units able to assemble into highly modular supramolecular architectures of arbitrary shapes (Figures 10.1, 10.4, 10.11 and 10.9). Besides classic cohesive Watson-Crick base pairing, the formation of longrange RNA-RNA interactions, such as loop-receptor or loop-loop interactions, offers a wide range of 4° intermolecular interfaces with various thermodynamic strengths to promote the cooperative assembly in the presence of divalent ions [35, 104, 176, 180] (Fig. 10.9). In the presence of magnesium, kissing loop motifs are more stable than RNA duplexes with identical sequences by two or three orders of magnitude [35, 180]. Moreover, the dynamic equilibrium of assembly through 4° RNA interfaces can be tuned over four to five orders of magnitude by adjusting the magnesium ion concentration and temperature. Thus, the hierarchical self-assembly of tectoRNAs can be monitored in a stepwise fashion to form architectures of increasing complexity [35], as there is a clear distinction between the energies involved in the formation of their 2°, 3° and 4° structures. In contrast to most DNA tiles, the formation of RNA tiles relies on the self-folding of single-stranded tectoRNAs that are characterized by well-defined 2° or/and 3° structures and 4° intermolecular interfaces.

0 0

Post a comment