Technological Uses of Molecular Flip-Flops

Molecular flip-flops such as the Fis flip-flop have at least two technological uses. First, binding equations for the flip-flop show that, compared to a single binding site, a dual-competitive site (in which both parts have the same binding energy) should have a doubled association constant [82]. This curious property may be useful in biological assays, because it increases the sensitivity without increasing the amount of bound protein [55].

Second, the flip-flop and other exclusionary binding site clusters may be used to construct a molecular computer since overlapping binding sites can provide Boolean logic [55]. Previous molecular-based computers have used DNA hybridization and PCR [87], and carbon monoxide on copper surfaces [88] among other methods. Steps have been made to evolve genetic circuits [89] and it has been demonstrated that the stability of gene networks can be increased by autoregulation [90] to provide sharp logical responses for `digital genetics'. Genetic networks have also been shown to provide distinct toggle switching [91,92] and Boolean logic [93]. In conjunction with these cell-sized chemical circuits, molecular flip-flops could be used to build molecular-scale circuits with sharp Boolean responses. A NOR gate can be constructed from a set of three binding sites, r1, a and r2, in which the middle site a is the binding site for a transcriptional activator A [55] (Fig. 8). When bound, A causes transcription of a downstream operon that can contain a gene for a signal such as GFP (Green Fluorescence Protein [94]) or a DNA binding protein for another part of the circuit. In the gate, r1 overlaps a and r2 overlaps a from the other side. As shown in Fig. 7, design of such constructs is facilitated by sequence walkers [10]. If either the R1 or the R2 binding protein exists in the cell or solution, then the A binding protein is excluded and the transcript is not expressed. Because this forms a NOR gate with amplification and fan-out at every step, any digital genetic circuit can be constructed, including complete computers [95,96,97,98]. This method can take advantage of the inherent precision of protein/DNA recognition to provide low error rates [99,100]. Unlike the gene-sized logic circuits previously described [93], which require about 3000 bases of DNA (1$R_{sequence} = 3.983 \pm 0.399$m), the functional component of these gates can be about 30 bases (10 nm) in length.