rapid-assembly-multi-gene-constructs-using-modular-golden-gate

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

State University of New York

Plants of the Digitalis genus contain a cocktail of cardenolides commonly prescribed to treat heart failure. Cardenolides in Digitalis extracts have been conventionally quantified by high-performance liquid chromatography yet the lack of structural information compounded with possible co-eluents renders this method insufficient for analyzing cardenolides in plants. The goal of this work is to structurally characterize cardiac glycosides in fresh-leaf extracts using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) that provides measured accurate mass. Fragmentation of cardenolides is featured by sequential loss of sugar units while the steroid aglycone moieties undergo stepwise elimination of hydroxyl groups, which distinguishes different aglycones. Using a reverse-phase LC column, the sequence of elution follows: diginatigenin→digoxigenin→gitoxigenin→gitaloxigenin→digitoxigenin for cardenolides with the same sugar units but different aglycones. A linear range of 0.8-500 ng ml has been achieved for digoxigenin, β-acetyldigoxin, and digitoxigenin with limits of detection ranging from 0.09 to 0.45 ngml. A total of seventeen cardenolides have been detected with lanatoside A, C, and E as major cardenolides in Digitalis lanata while seven have been found in Digitalis purpurea including purpurea glycoside A, B, and E. Surprisingly, glucodigifucoside in D. lanata and verodoxin and digitoxigenin fucoside in D. purpurea have also been found as major cardenolides. As the first MS/MS-based method developed for analyzing cardenolides in plant extracts, this method serves as a foundation for complete identification and accurate quantification of cardiac glycosides, a necessary step towards understanding the biosynthesis of cardenolide in plants.

Profiling and structural analysis of cardenolides in two species of Digitalis using liquid chromatography coupled with high-resolution mass spectrometry.

Cardiac glycosides, steroid derivatives extracted from the foxglove plants, have been used for the treatment of heart failure since the 18th century. A method based on liquid chromatography coupled with high-resolution tandem mass spectrometry (LC/MS) has been developed to characterize and quantify cardiac glycosides in fresh-leaf extracts of the foxglove () plants [1]. In this report, the fragmentation spectra of additional authentic standards of cardiac glycoside (digitoxigenin, digoxigenin, -acetyldigoxin) and cardenolides identified in the leaves of  () and  () were provided with high resolution. The exact mass of signature peaks for the aglycones and the sugar units of cardenolides were measured. This dataset is valuable to researchers interested in characterizing cardenolides in plants, or quantifying cardenolides in drug tablets, or studying cardenolide toxicities in animals. The fragmentation patterns of authentic cardenolide standards provided in these data can be used to validate relevant cardenolides in various biological samples and to infer chemical structures of unknown cardiac glycosides.

Zhen Q. Wang

Department of Biological Sciences,
University at Buffalo,
Department of Biological Sciences, University at Buffalo

State University of New York

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Zhen Q. Wang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences,University at Buffalo,Department of Biological Sciences, University at Buffalo

State University of New York

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Department of Biological Sciences,
University at Buffalo,
Department of Biological Sciences, University at Buffalo