Part I. Basic studies of the inhibitor DNA enzyme system. Part II. Universal nucleic acid translator.

摘要

In nature, the translation of genetic information (DNA/RNA) into cellular instructions (protein) is known as the central dogma of molecular biology. Not surprisingly then, the ability to translate, or rationally correlate conversion between two chemically unrelated species in vitro, is central to engineering the behavior of many chemical systems. For instance, the field of diagnostics is based upon the translation of small molecules, proteins, and nucleic acids into experimental observables such as fluorescent, luminescent, colorimetric or radioactive moieties. The research described herein focuses on two unique translation systems: first, the inhibitor DNA enzyme (IDE) system and, second, universal nucleic acid translators. The IDE system is inspired by naturally occurring intrasterically inhibited enzymes such as proteinases, kinases and phosphatases all of which act as "translators" by converting analyte binding events into biologically relevant signals. The IDE system mimics their intrasterically inhibited design by covalently modifying Cereus Neutral Protease (CNP) with an inhibitor attached via a piece of single stranded DNA. Due to the flexible nature of single stranded DNA and the high local concentration of the inhibitor, the attached inhibitor will spend the majority of the time in the active site of the enzyme preventing turnover of substrate. If a piece of single stranded DNA complementary to the tether region is added, however, the normally flexible tether region will rigidify upon double helix formation removing the inhibitor from the active site. The active enzyme can then turn over a fluorogenic peptide substrate creating an easily measurable signal. Herein we present basic studies describing the effect double stranded regions have on both the enzymatic activity and inhibition of this system. We also present an in-depth study that characterizes the scope and limitations of two emerging classes of universal nucleic acid translators, both of which utilize sequence directed DNA strand displacement as the underlying mechanism of translation. Translators are presented that convert several different biologically relevant diagnostic target sequences (Hepatitis C virus, avian influenza (H5N1), and the Smallpox virus) into a desired DNA output using both DNA, and in one case, genomic RNA from the Hepatitis C virus.