Difference between revisions of "Strand-Displacement"

Latest revision as of 21:36, 6 December 2007

The strand-displacement mechanism uses competitive binding of two identical nucleic acid sequences, the competing strand and the switching strand. It is based on rational design and results in the disruption or restoration of the hammerhead ribozyme as a result of restoration in the aptamer domain (Supplementary Information).

Competing strand

The competing strand is the nucleic acid sequence that is bound to the the general transmission region in the restored switch conformation in the presence of a ligand (Supplementary Information).

Switching strand

The switching strand is the nucleic acid sequence that is bound to the general transmission region in the disrupted switch conformation in the absense of a ligand (Supplementary Information).

ON and OFF Ribozyme Switches

Win and Smolke designed ON and OFF switches by the strand-displacement mechanism that allows either disruption or activation of the ribozyme catalytic core. The ON switch in Figure 2A begins with a ribozyme L2bulge1 that starts out in the active conformation with the aptamer unbound. When the aptamer is unbound, the catalytic core is not disrupted, which allows the ribozyme to self-cleave. The cleaving effect of the ribozyme causes down regulation of gene expression. By a simple nucleotide shift when the competing strand binds, the conformation of the aptamer where the ligand can bind changes, allowing theophilline (the ligand) to bind. When theophilline binds, the conformation of the catalytic core has a bulge in it, which prevents self-cleavage. Thus, the gene can now be up regulated. Ideally the two conformations of bound and unbound states would be constantly changing from one to the other, but when theophilline binds, it stays bound, which shifts the equilibrium so that more ribozymes end up in the ON state (Figure 2A). The researchers also show how the amount of gene expression is dependent on theophilline concentration and follows a dose response pattern (Figure 2C).
In much the same way, the OFF switch begins with a ribozyme that has a bulge and therefore is inactive and allows gene expression. When nucleotide shifting occurs, the aptamer is bound with theophilline and the ribozyme is allowed to cleave itself, resulting in the OFF state. Once again, the OFF switch has a dose response curve due to theophilline concentrations, which is important in determining gene regulation effects (Figure 1D). http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700002.jpeg

Figure 2. "Regulatory properties of the strand-displacement information transmission mechanism. The color scheme corresponds to that used in Fig. 1 with the following exceptions: switching strand, red; competing strand, green. (A) Gene expression ON ribozyme switch platform, L2bulge1. (B) Gene expression OFF ribozyme switch platform, L2bulgeOff1. (C and D) The theophylline-dependent gene-regulatory behavior of L2bulge1 (ON switch) (C), L2bulgeOff1 (OFF switch) (D), and L2Theo (nonswitch control). Gene-expression levels are reported in fold as defined in SI Text and were normalized to the expression levels in the absence of effector" (Win and Smolke, 2007). Image Permission Pending.

Tunability of Ribozyme Switches

Next, the investigators wanted to show that the ON and OFF switches created could be practical and applicable because they can be rationally designed to exhibit different levels of gene regulation. The swithes were created the same way as before except that they made many different aptamers that can be used to elicit differents responses of induction in fold of GFP due at 5 mM of theophylline (Figure 4B). The variations in inductions can be explained by the differences in energetics between the two states so that the aptamers with the least energy difference between the bound and unbound state have the highest induction Supplementary Information. http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700004.jpeg

Figure 3. "Tunability of the strand-displacement-based ribozyme switches. (A) Sequences targeted by the rational tuning strategies are indicated in the dashed boxes on the effector-bound conformations of L2bulge1 (ribozyme-inactive) and L2bulgeOff1 (ribozyme-active). (B and C) Regulatory activities of tuned strand-displacement-based ON (B) and OFF (C) ribozyme switches. Gene-regulatory effects of these switches at 5 mM theophylline are reported in fold induction for ON switches and fold repression for OFF switches relative to the expression levels in the absence of theophylline as described in Fig. 2" (Win and Smolke, 2007). Image Permission Pending.

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