Biomedical subjects

Karl Kossen

Publications and source records attributed to Karl Kossen.

4 recordsLinked to original sources

PubMed2004

High-throughput ribozyme-based assays for detection of viral nucleic acids.

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.

Base Sequence↗

PubMed2004

Monitoring protein modification with allosteric ribozymes.

An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.

Allosteric Regulation↗

PubMed2003

Zeptomole detection of a viral nucleic acid using a target-activated ribozyme.

We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.

Hepacivirus↗

PubMed2002

The carboxy-terminal domain of the DExDH protein YxiN is sufficient to confer specificity for 23S rRNA.

DE x DH proteins are believed to modulate the structures of RNAs and ribonucleoprotein complexes by disrupting RNA helices and RNA-protein interactions. All DE x DH proteins contain a two-domain catalytic core that enables their RNA-dependent ATPase and RNA helicase activities. The catalytic core may be flanked by ancillary domains that are proposed to confer substrate specificity and facilitate the unique functions of individual proteins. The Escherichia coli DE x DH protein DbpA and its Bacillus subtilis ortholog YxiN have similar 75aa carboxy-terminal domains, and both proteins are specifically targeted to 23S rRNA. Here we demonstrate that the carboxy-terminal domain of YxiN is sufficient to confer RNA specificity by characterizing a chimera in which this domain is appended to the core domains of E.coli SrmB, a DE x DH protein with no apparent substrate specificity. Both the RNA-dependent ATPase and RNA helicase activities of the chimera are specifically activated by 23S rRNA and abolished by sequence changes within hairpin 92, a critical recognition element for Y x iN. These data support a model in which the carboxy-terminal domain binds hairpin 92 to target the protein to 23S rRNA.

Adenosine Triphosphatases↗