Preparation of Duplex Sequencing Libraries for Archival Paraffin-Embedded Tissue Samples Using Single-Strand-Specific Nuclease P1.

DNA from formalin-fixed paraffin-embedded (FFPE) tissues, which are frequently utilized in cancer research, is significantly affected by chemical degradation. It was suggested that approaches that are based on duplex sequencing can significantly improve the accuracy of mutation detection in FFPE-derived DNA. However, the original duplex sequencing method cannot be utilized for the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, as FFPE DNA contains an excessive number of damaged bases, and these lesions are converted to false double-strand nucleotide substitutions during polymerase-driven DNA end repair process. To resolve this drawback, we replaced DNA polymerase by a single strand-specific nuclease P1. Nuclease P1 was shown to efficiently remove RNA from DNA preparations, to fragment the FFPE-derived DNA and to remove 5'/3'-overhangs. To assess the performance of duplex sequencing-based methods in FFPE-derived DNA, we constructed the Bottleneck Sequencing System (BotSeqS) libraries from five colorectal carcinomas (CRCs) using either DNA polymerase or nuclease P1. As expected, the number of identified mutations was approximately an order of magnitude higher in libraries prepared with DNA polymerase vs. nuclease P1 (626 ± 167/Mb vs. 75 ± 37/Mb, paired t-test p-value 0.003). Furthermore, the use of nuclease P1 but not polymerase-driven DNA end repair allowed a reliable discrimination between CRC tumors with and without hypermutator phenotypes. The utility of newly developed modification was validated in the collection of 17 CRCs and 5 adjacent normal tissues. Nuclease P1 can be recommended for the use in duplex sequencing library preparation from FFPE-derived DNA.

Heterologous expression and characterization of Penicillium citrinum nuclease P1 in Aspergillus niger and its application in the production of nucleotides.

Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg2+, Zn2+ and Cu2+, and inhibited by addition of Ca2+, Fe2+, Mn2+, Ni2+, Co2+, Mg2+, K+ and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5'- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.

Efficient Purification of Nuclease P1 from Penicillium citrinum Using Polyethylene Glycol/Disodium Guanosine Monophosphate Aqueous Two-Phase System.

Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5'-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum. Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa2) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa2 was also studied, and 95.1% of GMPNa2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine.

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2-5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%-110%, and the inter-day and intra-day precision were acceptable (RSD < 10%). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

32P-Postlabeling Analysis of DNA Adducts.

32P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA, and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts, and detection and quantification by means of their radioactive decay. Using 10 µg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 109-1010 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

Efficient nanobiocatalytic systems of nuclease P1 immobilized on PEG-NH2 modified graphene oxide: effects of interface property heterogeneity.

The use of graphene oxide (GO) nanosheets for functional enzyme support has attracted intensive interest owing to their unique planar structure and intriguing physical and chemical properties. However, the detailed effects of the interface properties of GO and its functionalized derivatives on active biomolecules are not well understood. We immobilize nuclease P1, a common industrial nucleic acid production enzyme, on pristine and amino poly(ethylene glycol) (PEG-NH2) modified GO nanosheets with interface property heterogeneity using two approaches, physical adsorption and chemical crosslinking. It is demonstrated that nuclease P1 could be stable immobilized on the surface of pristine GO by physical adsorption and on the edge of modified GO nanosheets by chemical crosslinking. The resultant loading capacity of nuclease P1 on pristine GO is as high as 6.45mg/mg as a consequence of strong electrostatic and hydrophobic interactions between the enzyme and carrier. However, it is determined that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on PEG-NH2-modified GO are obviously improved compared to those of the enzyme immobilized on pristine GO. The enhanced catalytic behavior demonstrates that GO and its derivatives have great potential in efficient biocatalytic systems.

Safety evaluation of nuclease P1 from Penicillium citrinum.

Nuclease P1 has been widely used in the food industry to enhance or create flavor. One commercial source of this enzyme is Penicillium citrinum, an anamorphic mesophilic fungus with a long history of safe use in Europe and Asia as a fermentation organism used in the production of ribonucleases. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including an in vitro Ames and chromosome aberration assay, an in vivo rat erythrocyte micronucleus assay and a 90-day oral toxicity study in rats were conducted. No mutagenic activity was observed in the Ames assay. Equivocal activity in the chromosome aberration assay was not replicated in the micronucleus assay at doses of up to 1007 mg total organic solids (TOS)/kg body weight (bw)/day. Following oral administration of nuclease P1 at dosages of 10.1, 101 or 1007 mg TOS/kg bw/day to Sprague-Dawley rats, no adverse effects on any study parameter were observed. The no-observed-adverse-effect level was considered to be 1007 mg TOS/kg bw/day. The results of the genotoxicity studies and subchronic rat study support the safe use in food production of nuclease P1 produced from P. citrinum.