Gene/Protein Disease Symptom Drug Enzyme Compound
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Herein, we describe a polymeric micellar nanoparticle capable of rendering nucleic acids resistant to nuclease digestion. This approach relies on utilizing DNA as the polar headgroup of a DNA-polymer amphiphile in order to assemble well-defined, discrete nanoparticles. Dense packing of DNA in the micelle corona allows for hybridization of complementary oligonucleotides while prohibiting enzymatic degradation. We demonstrate the preparation, purification, and characterization of the nanoparticles, then describe their resistance to treatment with endo- and exonucleases including snake-venom phosphodiesterase (SVP), a common, general DNA digestion enzyme.
ACS Nano 2013 Feb 26
PMID:Nuclease-resistant DNA via high-density packing in polymeric micellar nanoparticle coronas. 2337 79

Identification and quantification of chemical DNA modifications provide essential information on genomic DNA changes, for example, epigenetic modifications and abnormal DNA lesions. In this vein, it requires to digest genomic DNA strands into single nucleosides, facilitating the mass spectrometry analysis. However, rapid digestion of such supramacromolecule DNA of several millions Daltons (molecular weight) into single nucleosides remains very challenging. Here, we constructed an immobilized benzonase capillary bioreactor and further tandemly coupled with immobilized snake venom phosphodiesterase and alkaline phosphatase capillary bioreactor to form a novel three-enzyme cascade bioreactor (BenzoSAC bioreactor). In these constructions, the chosen enzymes were immobilized onto synthetic porous capillary silica monoliths. With the tailor-made porous structure and high immobilized capacity and high digestion rate of benzonase, genomic DNA of >99.5% can be digested into single nucleosides within only 10 min when passing through the BenzoSAC bioreactor by microinjection pump. In contrast, traditional digestion requires 8-24 h. By offline coupling this benzoSAC bioreactor with liquid chromatography-tandem mass spectrometry, we detected 5-hydroxymethylcytosine, a major oxidation product of the epigenetically crucial 5-methylcytosine, in genomic DNA isolated from ladder cancer (T24) cells. The newly synthesized BenzoSAC bioreactor and the proposed mass spectrometry detection are promising for fast identification and analysis of structural modifications in DNA.
ACS Appl Mater Interfaces 2018 Jul 05
PMID:Multienzyme Cascade Bioreactor for a 10 min Digestion of Genomic DNA into Single Nucleosides and Quantitative Detection of Structural DNA Modifications in Cellular Genomic DNA. 2988 39

The metazoan second messenger 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is a cyclic dinucleotide (CDN) that induces secretion of type I interferons and activates the immune system and has thus attracted significant interest as a vaccine adjuvant or immunotherapeutic. CDN bisphosphorothioates are of particular interest because of their increased hydrolytic stability and improved cell activities. In our work with CDN bisphosphorothioates, we sought a method for systematic determination of the absolute stereochemistry of their phosphorothioate stereocenters. A novel biocatalytic method employing snake venom phosphodiesterase (svPDE) and nP1 has been developed and successfully applied to stereochemistry determination of 2'3'-cGAMP bisphosphorothioates. This method unambiguously assigned the phosphorothioate stereochemistry of four diastereomers of 2'3'-cGAMP bisphosphorothioate by analyzing distinct hydrolysis patterns of the bisphosphorothioate diastereomers upon incubation with svPDE and nP1. Furthermore, the regiospecificity as well as stereospecificity of both svPDE and nP1 toward 2'3'-cGAMP bisphosphorothioate has been elucidated.
ACS Omega 2020 Jun 16
PMID:Novel Applications of Biocatalysis to Stereochemistry Determination of 2'3'-cGAMP Bisphosphorothioate (2'3'-cGSASMP). 3256 85