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Chen N.G.,Sci-Tec, Inc. | Golovlev V.,Sci-Tec, Inc.
Molecular Informatics | Year: 2013

Little published literature exists on the 881 bit structural keys used by PubChem for categorizing and comparing the compounds present in its database. We characterized these structural keys by examining their frequencies of occurrence within the PubChem compound database. In addition, bit dependencies, defined as the universal presence of a bit given the presence of another, were determined. We show that the vast majority of bits are rarely set and that substantial numbers of dependencies exist. A comparison of similarity searches with five United States Food and Drug Administration approved drugs as reference compounds using the full structural keys versus a variant in which all dependent bits were removed was performed using the Tanimoto coefficient. These bit dependencies not only affect similarity scores, but also alter the compounds returned in similarity searching. Judicious selection of bits is needed to maintain sufficient ability to differentiate related compounds. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Sun Y.,Sci-Tec, Inc. | Gregory K.J.,Sci-Tec, Inc. | Chen N.G.,Sci-Tec, Inc. | Golovlev V.,Sci-Tec, Inc.
Analytical Biochemistry | Year: 2012

A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay uses miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated high cross-platform consistency between this assay and the TaqMan real-time polymerase chain reaction (PCR) assay with R2 = 0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than 1 h. Compared with the real-time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis. © 2012 Elsevier Inc. All rights reserved.


Chen N.G.,Sci-Tec, Inc. | Gregory K.,Sci-Tec, Inc. | Sun Y.,Sci-Tec, Inc. | Golovlev V.,Sci-Tec, Inc.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2011

The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation. © 2011 Elsevier B.V. All rights reserved.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.89K | Year: 2010

DESCRIPTION (provided by applicant): The goal of this SBIR Phase I project is the development of a new drug discovery platform for screening new inhibitors of viral polymerases and for studying mechanisms of action of the prospective pharmaceutical compounds. The main innovation in this project is the proposed concept of a multi-enzyme assay, which is able to measure efficiency and identify the mechanism of action of a viral polymerase inhibitor in a single experiment using a small reaction volume in realtime detection mode using widely available commercial instruments (luminometers). The proposed platform is superior over the existing methods in that it substantially reduces the cost of reagents, reduces the assay time from days to minutes and is adaptable to high-throughput format for application in large and small scale drug discovery projects. The proposed SBIR project is in response to the NIH call for new approaches aimed at reducing costs and increasing speed of preclinical drug development. PUBLIC HEALTH RELEVANCE: Antiviral therapy represents an important frontier for development of potential lifesaving products. Worldwide, millions of people suffer from viral infections each year. Approximately 360 million people worldwide suffer from hepatitis B virus, the major cause of liver cancer. Worldwide more than 170 million people are infected with hepatitis C, the leading cause of acute liver inflammation and liver cancer. Around the world more than 42 million people are infected with lifethreatening human immunodeficiency virus (HIV), including more than 3 million children. To address the growing medical needs, there is a steady increase in the number of antiviral compounds in development. The proposed SBIR project will create a knowledge base and establish a new technology, which reduces the cost and time of developing pharmaceutical antiviral products with a large health impact in the USA and around the world.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 760.17K | Year: 2010

DESCRIPTION (provided by applicant): The goal of this project is to develop a new highly sensitive and cost effective RNA Enzymatic Luminescence Assay (qELA) for high-throughput detection and quantification of microRNA in biological samples. The assay implements the same detection concept known from pyrosequencing, yet expanding pyrosequencing detection methodology for highly sensitive and accurate quantification of small RNA molecules. The proposed assay can match the performance of the real time quantitative PCR technique (rt-qPCR) in many applications and is simpler and more cost effective. The technology can be implemented in a number of commercial assays for application in life science research, drug discovery, and clinical diagnosis. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a new high performance cost effective bioluminescence assay for microRNA analysis. The proposed assay provides technologically advanced and significantly less expensive alternative for real time polymerase chain reaction technology, which currently dominates in the market. The proposed bioluminescent assay is universal and can be used in various applications in life science research, drug discovery, clinical diagnosis by 1,800 clinical laboratories and thousands of life science research laboratories in US.


PubMed | Sci-Tec, Inc.
Type: Journal Article | Journal: Analytical biochemistry | Year: 2012

A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay uses miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated high cross-platform consistency between this assay and the TaqMan real-time polymerase chain reaction (PCR) assay with R(2)=0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than 1h. Compared with the real-time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.

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