RNA and DNA are highly programmable polymers due to their ability to form specific Watson-Crick base pairing; a property that can be exploited to create well — defined 2D and 3D structures (Rothemund 2006; Seeman 2010). These structures can be thermodynamically stable, and formed via spontaneous self-assembly, a process that requires no catalytic co-factors. In addition to Watson-Crick base pairing, an increasing number of useful tertiary RNA motifs are being discovered in nature. These simple motifs, such as three-way junctions and interacting loops, can serve as diverse building blocks for construction of higher-order structures with increasing complexity.

Single-stranded RNA can be continuously expressed at high levels in live cells, and thus offers the opportunity to program cells to assemble designer nanostructures for specific cellular functions. One potential application of such intracellular nanostructures that is particularly relevant to algal biofuel optimization is the construction of RNA scaffolds (Fig. 8.3). The aim of such RNA scaffolds is to provide a spatially organized docking station where different proteins can ‘park’ at pre-determined distances and permutations. This higher level of organization can be designed to bring together a specific set of enzymes and organize them at fixed proximity and orientations. The configuration could be used to limit the diffusion of intermediate substrates, hence efficiently channel substrates to final products over several enzymatic steps leading to increased yields from sequential metabolic or cellular reactions (Dueber et al. 2009). An example of this utility in bacteria is demonstrated in a recent study where RNAs are engineered and expressed as 2D scaffolds to spatially organize enzymes that effectively lead to increased reaction output by 48-fold (Delebecque et al. 2011). This points to the possibility that RNA scaffolds could be used to engineer biosynthetic pathways to maximize algal biofuel production.

In general, constructing RNA scaffolds involves the following steps (Delebecque et al. 2012). The first is to design the overall RNA secondary structure with minimal predicted free energy. RNA folding software such as RNA Designer, mfold, and NUPACK can be used to design RNA sequences that fold into desired shapes. Considerations generally include the GC percentage, removal of problematic sequences that may cause alternative folding, and removal of motifs such as splice sites and poly(A) signals that may be processed by cells. The next step is choosing appropriate aptamers that allow the coupling of proteins to the RNA scaffolds. Aptamers are folded RNA structures that function as receptors with specific ligand binding properties. Selective aptamers can be incorporated into the RNA scaffold as the docking sites. Aptamers with high binding affinity and specificity are required to

Fig. 8.3 Conceptual illustration of optimizing algal metabolism using RNA scaffolds. RNA scaffold constructs are RNA structures introduced into algae by genetic transformation to form and guide higher order assembly of metabolic enzymes. These composite structures can optimize bioproduction of high value metabolites

achieve good protein docking interactions. RNA sequences can be designed to fold into either discrete self-standing scaffolds, or with additional polymerization between discrete scaffolds to form a more complex structure (Fig. 8.3). In a discrete RNA scaffold, the aptamer binding sites are located within the same RNA molecule separated by spacer sequences. In a polymerizing RNA scaffold, each discrete RNA within the polymer may carry a different aptamer to capture the desired enzyme. The resulting polymer structure is a higher-level one, two or even three-dimensional scaffold with aptamers placed in specific locations. In this case, secondary inter­actions, kinetic considerations, and non-canonical interactions among the
components of the RNA scaffold must all be considered in the secondary structure design process. Common examples of polymerizing RNA scaffolds are nanotubes and two-dimensional sheets. In such complicated cases, designing the RNA scaffold to be an assembly of modular parts might make the process simpler. Another strategy for simplifying polymerizing RNA scaffold design and assembly is the reliance on palindromic sequences as a design strategy. Palindromic sequences create a symmetrical structure that simplifies scaffold design and minimizes the interactions required to assemble the overall structure. Obtaining a desired RNA scaffold structure usually requires an iterative process that relies on experimental verifications. Finally, a possibly helpful design consideration in RNA scaffold sequences is the incorporation of restriction sites within the different relevant sequences to allow for interchangeability and modifications within the RNA scaffold structure.

The use of RNA scaffolds for the purpose of increasing metabolic reactions hinges on the ability to precisely engineer higher-order scaffolds in the complex cellular environment. There remain several challenges that may limit the general application of RNA nanostructure in the cellular environment. First, thousands of cellular RNAs and proteins that are present in cells may non-specifically interact with individual RNA building blocks and prevent the formation of higher-order structures. Second, RNA scaffolds may cause cellular stress and disrupt cellular functions leading to general toxicity. Additional concerns include the stability and half-life of RNA scaffolds, and whether the scaffolds could be targeted to the appropriate cellular compartment such as cytoplasm vs. nucleus. Nevertheless, the ever-increasing number of useful RNA motifs and knowledge of RNA biology at our disposal may help to solve these issues in the near term.