超声波提取HPLC-UV法测定人工蛹虫草中8种核苷类物质

蛹虫草中核苷及其代谢化合物的分析检测对于蛹虫草的生理和药理研究是非常重要的。该文采用超声波辅助提取HPLC-UV法,以反相DIAMONSIL C1(8250mm×460mm,5μm)为色谱柱,以水(含0.1%乙酸)和甲醇混合液为流动相进行梯度洗脱,同时测定了人工蛹虫草中的尿嘧啶、次黄嘌呤、鸟嘌呤、胸腺嘧啶、肌苷、鸟苷、腺苷和虫草素等8种化合物。结果表明,超声波提取的最优条件为提取溶剂为甲醇,提取温度25℃,提取时间15min,超声功率100W,料液比1:100,在最优的超声提取和色谱分离条件下,8种核苷类化合物在检测范围内的线性关系较好(r2>0.9995),检出限和定量限最低可达到16.9ng/mL和56.4ng/mL,日内和日间精密度(RSD)分别小于4.1%和4.6%,加样回收率为81.9%~93.7%。此法可用于冬虫夏草及其代用品中核苷类化合物的分析检测。     

Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine

5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase β. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.     

Synthesis and structural characterization of 2'-fluoro-α-L-RNA-modified oligonucleotides

We describe the synthesis and binding properties of oligonucleotides that contain one or more 2'-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2'-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2'-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2'-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson--Crick rules. The 2'-fluoro-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2'-fluoro monomer adopts a C3'-endo/C2'-exo sugar pucker. Molecular modeling of modified duplexes show that the 2'-fluoro-α-L-RNA nucleotides partake in Watson--Crick base pairing and nucleobase stacking when incorporated in duplexes while the unnatural α-L-ribo configured geometry of the sugar is absorbed by changes in the sugar-phosphate backbone torsion angles. The duplex behavior of our new nucleotide follows that of α-L-LNA, by and large.     

Hydration of AMP and ATP Molecules in Aqueous Solution and Solid Films

Water enables life and plays a critical role in biology. Considered as a versatile and adaptive component of the cell, water engages a wide range of biomolecular interactions. An organism can exist and function only if its self-assembled molecular structures are hydrated. It was shown recently that switching of AMP/ATP binding to the insulin-independent glucose transporter Human Erythrocyte Glucose Transport Protein (GLUT1) may greatly influence the ratio of bulk and bound water during regulation of glucose uptake by red blood cells. In this paper, we present the results on the hydration properties of AMP/ATP obtained by means of dielectric spectroscopy in aqueous solution and for fully ionized forms in solid amorphous films with the help of gravimetric studies.     

In Vivo Assembly and Expression of DNA Containing Non-canonical Bases in the Yeast Saccharomyces cerevisiae

Chemically modified nucleic acids are of utmost interest in synthetic biology for creating a regulable and sophisticated synthetic system with tailor-made properties. Implanting chemically modified nucleic acids in microorganisms might serve biotechnological applications, while using them in human cells might lead to new advanced medicines. Previously, we reported that a fully modified DNA sequence (called DZA) composed of the four base-modified nucleotides – 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine and 5-fluorocytosine – could function as a genetic template in prokaryotic cells, Escherichia coli. Here, we report the synthesis of long, partially, or fully modified DZA fragments that encode the yeast-enhanced red fluorescent protein (yEmRFP). The DZA sequences were directly introduced in the genome of the eukaryotic cells, Saccharomyces cerevisiae, via the yeast natural homologous recombination machinery. The simple and straightforward DZA cloning strategy reported here might be of interest to scientists working in the field of xenobiology in yeast.     

A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: in vivo characterization and structure/function relationships

Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity.     

Current Approaches for RNA Labeling in Vitro and in Cells Based on Click Reactions

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.     

Fluorescent analysis of translesion DNA synthesis by using a novel, non-natural nucleotide analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2-APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real-time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function.