Simple identification of two causes of noise in an aptazyme system by monitoring cell-free transcription.

Abstract

Aptazymes are artificially synthesized ribozymes that catalyze reactions in response to ligand binding. Certain types of aptazymes can be utilized as RNA-based regulators of gene expression. These aptazymes contain a sequestered ribosome-binding site (rbs) and release the rbs through self-cleavage in response to ligand binding, inducing the expression of the downstream gene. One of the most important properties of aptazymes as gene expression regulators is their signal-to-noise ratio (S/N ratio), the ratio of target expression in the presence of ligand to that in the absence of ligand. One strategy to improve the S/N ratio is to decrease the noise (expression in the absence of ligand) due to leaky translation without rbs release or ligand-independent rbs release. In this chapter, we describe an easy method to identify the main cause of noise using a cell-free reconstituted transcription-translation system, an ideal platform for the quantitative understanding of biochemical reactions because researchers can strictly control the experimental conditions and the concentrations of all components. This knowledge would be useful for designing aptazymes with high S/N ratios.