Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools

Abstract deoxynucleoside triphosphate (dNTPs) are the reduced nucleotides used as the building blocks and energy source for deoxyribonucleic acid (DNA) replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways that are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understanding the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilizing the current state-of-the-art polymerase method, modified to a solid-phase setting and optimized for larger scale measurements.

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Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools

10.1101/270454 ◽

2018 ◽

Author(s):

Juan Cruz Landoni ◽

Liya Wang ◽

Anu Suomalainen

Keyword(s):

Dna Replication ◽

Cell Fate ◽

State Of The Art ◽

Solid Phase ◽

Building Blocks ◽

Current State ◽

Cell Processes ◽

Deoxynucleoside Triphosphate ◽

Phase Setting ◽

Solid Phase Assay

Deoxyribonucleoside triphosphates (dNTPs) are the reduced nucleotides used as the building blocks and energy source for DNA replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understand the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilising the current state-of-the-art polymerase method, modified to a solid-phase setting and optimised for larger scale measurements.

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DNA functionalization by dynamic chemistry

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.12.203 ◽

2016 ◽

Vol 12 ◽

pp. 2136-2144 ◽

Cited By ~ 1

Author(s):

Zeynep Kanlidere ◽

Oleg Jochim ◽

Marta Cal ◽

Ulf Diederichsen

Keyword(s):

Deoxyribonucleic Acid ◽

Solid Phase ◽

Building Blocks ◽

Thermodynamic Control ◽

Dna Templates ◽

Phase Synthesis ◽

Dynamic Combinatorial Chemistry ◽

Dna Oligonucleotides ◽

Reversible Reactions ◽

Modified Nucleobases

Dynamic combinatorial chemistry (DCC) is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80%) was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

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Development of Strategies for Glycopeptide Synthesis: An Overview on the Glycosidic Linkage

Current Organic Chemistry ◽

10.2174/1385272824999200701121037 ◽

2020 ◽

Vol 24 (21) ◽

pp. 2475-2497

Author(s):

Andrea Verónica Rodríguez-Mayor ◽

German Jesid Peralta-Camacho ◽

Karen Johanna Cárdenas-Martínez ◽

Javier Eduardo García-Castañeda

Keyword(s):

Chemical Synthesis ◽

Solid Phase ◽

Building Blocks ◽

Heterogeneous Environments ◽

Phase Synthesis ◽

Low Concentrations ◽

Biological Sources ◽

Synthetic Routes ◽

The Impact ◽

Potential Use

Glycoproteins and glycopeptides are an interesting focus of research, because of their potential use as therapeutic agents, since they are related to carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions, which are commonly involved in biological processes. It has been established that natural glycoconjugates could be an important source of templates for the design and development of molecules with therapeutic applications. However, isolating large quantities of glycoconjugates from biological sources with the required purity is extremely complex, because these molecules are found in heterogeneous environments and in very low concentrations. As an alternative to solving this problem, the chemical synthesis of glycoconjugates has been developed. In this context, several methods for the synthesis of glycopeptides in solution and/or solid-phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for both solution and solid-phase synthesis. The synthetic viability of glycoconjugates is a critical parameter for allowing their use as drugs to mitigate the impact of microbial resistance and/or cancer. However, the chemical synthesis of glycoconjugates is a challenge, because these molecules possess multiple reaction sites and have a very specific stereochemistry. Therefore, it is necessary to design and implement synthetic routes, which may involve various protection schemes but can be stereoselective, environmentally friendly, and high-yielding. This review focuses on glycopeptide synthesis by recapitulating the progress made over the last 15 years.

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Fully Automated Parallel Oligonucleotide Synthesizer

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20011299 ◽

2001 ◽

Vol 66 (8) ◽

pp. 1299-1314 ◽

Cited By ~ 14

Author(s):

Michal Lebl ◽

Christine Burger ◽

Brett Ellman ◽

David Heiner ◽

Georges Ibrahim ◽

...

Keyword(s):

Solid Phase ◽

Building Blocks ◽

Solid Support ◽

Continuous Operation ◽

Gear Pump ◽

Oligonucleotide Synthesis ◽

Center Of Rotation ◽

Microtiter Plates ◽

The Individual ◽

Design And Construction

Design and construction of automated synthesizers using the tilted plate centrifugation technology is described. Wash solutions and reagents common to all synthesized species are delivered automatically through a 96-channel distributor connected to a gear pump through two four-port selector valves. Building blocks and other specific reagents are delivered automatically through banks of solenoid valves, positioned over the individual wells of the microtiterplate. These instruments have the following capabilities: Parallel solid-phase oligonucleotide synthesis in the wells of polypropylene microtiter plates, which are slightly tilted down towards the center of rotation, thus generating a pocket in each well, in which the solid support is collected during centrifugation, while the liquid is expelled from the wells. Eight microtiterplates are processed simultaneously, providing thus a synthesizer with a capacity of 768 parallel syntheses. The instruments are capable of unattended continuous operation, providing thus a capacity of over two millions 20-mer oligonucleotides in a year.

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Synthesis and Self-assembly of Amphiphilic Precision Glycomacromolecules

Polymer Chemistry ◽

10.1039/d1py00422k ◽

2021 ◽

Author(s):

Alexander Banger ◽

Julian Sindram ◽

Marius Otten ◽

Jessica Kania ◽

Alexander Strzelczyk ◽

...

Keyword(s):

Self Assembly ◽

Solid Phase ◽

Building Blocks ◽

Polymer Synthesis

We present the synthesis of so called amphiphilic glycomacromolecules (APGs) by using solid-phase polymer synthesis. Based on tailor made building blocks, monosdisperse APGs with varying compositions are synthesized, introducing carbohydrate...

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Future trends in diagnosis using laboratory-on-a-chip technologies

Parasitology ◽

10.1017/s0031182099004126 ◽

1999 ◽

Vol 117 (7) ◽

pp. 191-203 ◽

Cited By ~ 16

Author(s):

M. S. TALARY ◽

J. P. H. BURT ◽

R. PETHIG

Keyword(s):

Gene Expression ◽

Lower Cost ◽

Building Blocks ◽

Cancer Diagnostics ◽

Future Trends ◽

Biotechnological Applications ◽

Environmental Testing ◽

Current State ◽

Microelectronic Fabrication ◽

Wide Range

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

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