Abstract
Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications.
Highlights
Nucleic acid binding species have emerged as a powerful tool for molecular recognition, and have begun to be widely adapted as biosensors, in drug-delivery systems, and as regulatory elements that control gene expression [1,2,3,4]
While there are numerous empirical examples of aptazymes operating as biosensors and regulatory elements, quantitative analyses of aptazyme performance and the development of design principles for aptazymes have seldom been attempted and are largely incomplete [10,16]
While it was concluded that ‘‘a design that is biased toward forming the disrupted-aptamer conformation will generally increase the dynamic range ...(but) require higher ligand concentrations to modulate protein level,’’ the more useful quantitative relationship between dynamic range of activity and ligand sensitivity that should enable rational design was not described
Summary
Nucleic acid binding species (aptamers) have emerged as a powerful tool for molecular recognition, and have begun to be widely adapted as biosensors, in drug-delivery systems, and as regulatory elements that control gene expression [1,2,3,4]. When designing aptazymes it is important to consider the fold-activation of the cleavage rate constant when ligand concentration increases from 0 to its highest possible concentration. We denote this foldactivation as gR, Realistic and formally define it as: Realistic. When designing ligand-inhibited aptazymes as in vitro biosensors, it is always beneficial to choose a low cleavage tendency as long as kapp(max) is still readily detectable For both ligand-activated and ligand-inhibited aptazymes there are trade-offs between ligand sensitivity and the dynamic range of activity, reflected by equations (10) and (15), respectively. It should be noted that self-cleaving HHRzs placed within the 59 UTR can abet even stronger inhibition of gene expression [20], but such a model would be inherently more challenging because it would have to take into account the continuous scanning by the pre-initiation complex
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