Obelin mutants as reporters in bioluminescent dual-analyte binding assay

Author Posting. © Royal Society of Chemistry, 2013. This article is posted here by permission of RSC Publishing for personal use, not for redistribution. The definitive version was published in Analytical Methods 5 (2013): 636-640, doi:10.1039/C2AY25976A.Two obelin mutants distinctly different in the color of bioluminescence were successfully applied to simultaneous detection of two analytes in a single well. The flash-type signals were triggered by single injection of Ca2+ and were discriminated by the spectral and time resolutions. The technique was developed in a high-throughput format, and applied to simultaneous immunoassay of two gonadotropic hormones – luteinizing (lutropin or hLH) and follicle stimulating (hFSH) – in clinical sera, and to simultaneous detection of two gene allelic variants at single nucleotide polymorphism genotyping of the human F5 gene encoding factor V Leiden polymorphism 1691 G/A (R506Q). A considerable number of samples were investigated and the results obtained were in good correlation with those obtained by using traditional techniques.This work was supported by the Program of the Government of Russian Federation “Measures to attract leading scientists to Russian educational institutions” (grant no. 11. G34.31.058).2013-11-2

Про правову природу батьківських прав

Красицька Л. В. Про правову природу батьківських прав / Л. В. Красицька // Актуальні проблеми держави і права : зб. наук. пр. / редкол.: С. В. Ківалов (голов. ред.), В. М. Дрьомін (заст. голов. ред.), Ю. П. Аленін [та ін.] ; МОНмолодьспорт України, НУ «ОЮА». – Одеса : Юрид. л-ра, 2012. – Вип. 66. - С. 172-178.У статті розглядаються положення щодо правової природи батьківських прав, яка може бути визначена тільки стосовно окремих видів батьківських прав. Робиться висновок, що всі батьківські права є відносними з різним характером захисту, немайнові батьківські права є особистими

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases

Bioluminescent aptamer-based solid-phase microassay to detect lung tumor cells in plasma

Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5' end of both aptamers carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p<0.001). The area under ROC curve with the value of 0.901 distinguishes well between the two groups under investigation

Development and characterization of novel 2′-F-RNA aptamers specific to human total and glycated hemoglobins

Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c. The developed 2'-F-RNA aptamers have shown their applicability for detection of total and glycated hemoglobin in one sample using the solid-phase sandwich assay

Генетически модифицированные целентеразин-зависимые люциферазы как репортеры для анализа in vitro

Among the most important tasks of modern biotechnology is the development of new analytical techniques suitable for highly sensitive and specific detection of diagnostically important targets rapidly expanding their range with the progress in biomedical science. In this view, particular attention is paid to elaboration of methods based on photosensitive technologies (fluorescent, chemiluminescent, etc.). The studies on bioluminescent systems of various luminous organisms are being carried out in the laboratory of photobiology of the Institute of Biophysics SB RAS for more than 30 years. The genes of many light-emitting proteins, photoproteins and luciferases, were cloned. The recombinant protein analogues were obtained, their spatial structures were determined and the mechanisms of functioning were proposed. With site-directed mutagenesis applied, the luciferase variants with unique beneficial properties were created. Highly sensitive analytical systems for diagnosing a number of biologically active compounds – hormones, oncogenes, infectious agents, etc. were developed on the basis of bioluminescent reactions of these proteins. The paper presents the recent years’ results of these studies. These are the method of simultaneous detection of two targets in one sample on the basis of color variants of Ca2+-regulated photoprotein obelin and the method for rapid and sensitive detection of tick-borne encephalitis virus based on the unique biospecific fusion protein of Renilla luciferase. The research results testify that the proposed analytical systems are competitive and applicable in modern medical laboratoryОдна из важнейших задач современной биотехнологии – создание новых методов анализа, пригодных для высокочувствительного и специфичного выявления диагностически важных мишеней, спектр которых стремительно расширяется с развитием биомедицинской науки. При этом особое внимание уделяется разработке методов, основанных на фотосенсорных технологиях (флуоресцентных, хемилюминесцентных и пр.). В лаборатории фотобиологии Института биофизики СО РАН более 30 лет исследуются биолюминесцентные системы различных светящихся организмов. Клонированы гены многих светоизлучающих белков – фотопротеинов и люцифераз, получены их рекомбинантные аналоги, установлены пространственные структуры и предложены механизмы их функционирования. Сайт-направленным мутагенезом созданы варианты люцифераз с уникальными полезными свойствами. На основе биолюминесцентной реакции этих белков разработаны высокочувствительные аналитические системы для диагностики ряда биологически активных соединений – гормонов, онкогенов, инфекционных агентов и т.д. В настоящей работе представлены результаты этих исследований последних лет. Среди них – способ одновременного выявления двух мишеней в одном образце на основе цветных вариантов Са2+‑регулируемого фотопротеина обелина, а также способ быстрого и чувствительного выявления вируса клещевого энцефалита на основе уникального биоспецифического гибридного белка люциферазы Renilla. Результаты исследований свидетельствуют о конкурентоспособности предлагаемых аналитических систем и перспективности их применения в условиях современной медицинской лаборатори

Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering &ldquo;character&rdquo;

Ca²⁺-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering “character”

The Ca2+-Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation

Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer–target complexes during design and development of the specific aptamers. The assay involves Ca2+-regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay