Erasmus Schneider

A structural analysis of in vitro catalytic activities of hammerhead ribozymes

BackgroundRibozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes.ResultsWe employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and their catalytic activities were measured in vitro. We found that target disruption energy owing to the alteration of the local target structure necessary for ribozyme binding, and the total energy change of the ribozyme-target hybridization, are two significant parameters for prediction of ribozyme activity. Importantly, target disruption energy is the major contributor to the predictability of ribozyme activity by the total energy change. Furthermore, for a target-site specific ribozyme, incorrect folding of the catalytic core, or interactions involving the two binding arms and the end sequences of the catalytic core, can have detrimental effects on ribozyme activity.ConclusionThe findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. Tools implementing these rules are available from the Sribo module and the Srna module of the Sfold program available through Web server at http://sfold.wadsworth.org.

Measurement of Unconjugated Estriol in Serum by Liquid Chromatography–Tandem Mass Spectrometry and Assessment of the Accuracy of Chemiluminescent Immunoassays

BACKGROUND Unconjugated estriol (uE3) is routinely analyzed in clinical laboratories as risk assessment for Down syndrome. Immunoassays of various types are the most commonly used methods. The accuracies of RIAs and ELISAs for uE3 have been questioned, and to date there have been no independent studies investigating the accuracy of the relatively new chemiluminescent immunoassays. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for uE3 measurements in serum. METHODS Serum samples from patients in the second trimester of pregnancy were used, and uE3 concentrations were measured by LC-MS/MS and the Beckman Coulter Access® 2 and Siemens IMMULITE 2000 automatic chemiluminescent immunoassay analyzers. RESULTS The LC-MS/MS method was validated and showed limit of detection 0.05 ng/mL; limit of quantification 0.2 ng/mL; linearity of response to 32 ng/mL; total imprecision of 16.2%, 10.4%, and 8.2% for uE3 at 1.10, 4.18, and 8.32 ng/mL, respectively; and analytical recoveries of 95.9%-104.2%. ANOVA of the correlation for LC-MS/MS results vs chemiluminescent immunoassays results showed R(2) = 0.9678 (Access 2 = 0.9305 LC-MS/MS + 0.2177, Sy|x = 0.1786, P < 0.0001), and R(2) = 0.9663 (IMMULITE 2000 = 0.8849 LC-MS/MS - 0.0403, Sy|x = 0.1738, P < 0.0001). Bland-Altman plots of uE3 results revealed concentration-dependent immunoassay biases. Mock risk analysis for Down syndrome showed no apparent difference in the risk assessment outcomes if the adjusted method-specific multiples of the median were used, and the assay imprecision was <10% CV. CONCLUSIONS Standardization of immunoassay methods for uE3 analysis is needed to improve the accuracy of the measurements.

Societal challenges of precision medicine : Bringing order to chaos

The increasing number of drugs targeting specific proteins implicated in tumourigenesis and the commercial promotion of relatively affordable genome-wide analyses has led to an increasing expectation among patients with cancer that they can now receive effective personalised treatment based on the often complex genomic signature of their tumour. For such approaches to work in routine practice, the development of correspondingly complex biomarker assays through an appropriate and rigorous regulatory framework will be required. It is becoming increasingly evident that a re-engineering of clinical research is necessary so that regulatory considerations and procedures facilitate the efficient translation of these required biomarker assays from the discovery setting through to clinical application. This article discusses the practical requirements and challenges of developing such new precision medicine strategies, based on leveraging complex genomic profiles, as discussed at the Innovation and Biomarkers in Cancer Drug Development meeting (8th-9th September 2016, Brussels, Belgium).

Detection of B- and T-cell-specific gene rearrangements in 13 cell lines and 50 clinical specimens using the BIOMED-2 and the original InVivoScribe primers

The diagnosis of clonality by molecular techniques generally depends on the detection of unique molecular events such as specific gene rearrangements in a small percentage of malignant cells in the...