Nucleoside/Nucleotide Analysis Service

What is Nucleoside/Nucleotide?

Nucleosides and nucleotides are essential components of nucleic acids, which are the basic building blocks of RNA and DNA. A nucleoside consists of a purine (A, G) or pyrimidine (C, T, U) nitrogenous base and a five-carbon sugar (ribose or deoxyribose) bonded together. Though the nucleotide normally refers nucleoside monophosphate, now nucleoside diphosphate or nucleoside triphosphate are also belonged to nucleotides. In other words, a nucleotide consists of a nucleobase, a pentose, and a phosphate groups. The presence of phosphate groups distinguishes nucleotides from nucleosides. Importantly, they play crucial roles in storing and transmitting genetic information and are involved in almost all biochemical reaction processes in organisms, including energy transfer (e.g., ATP), signaling pathways, and enzymatic reactions. The determination of nucleosides and nucleotides is widely used in various fields such as biochemistry, medicine, food, and metabolomics. Many clinically used antiviral drugs are nucleoside or nucleotide analog drugs.

Figure 1. The structure of Nucleoside and Nucleotide.

Nucleosides and nucleotides are distributed along with nucleic acids in various organs, tissues, and cells in organisms, and participate in basic life activities such as inheritance, development, and growth of organisms. There are also considerable amounts of Nucleosides and nucleotides existing in free form in organisms. For example, adenosine triphosphate plays a major role in cellular energy metabolism. Energy release and absorption in the body are mainly reflected by the production and consumption of ATP. In addition, uridine triphosphate, cytidine triphosphate and guanosine triphosphate are also sources of energy in the anabolism of some substances. Adenylate is also a component of certain coenzymes, such as coenzymes I, II and coenzyme A. In living organisms, nucleotides can be synthesized from some simple compounds. These synthetic raw materials include aspartic acid, glycine, glutamine, one-carbon units, and CO₂, etc. The catabolism of purine nucleotides in the body can produce uric acid, and the decomposition of pyrimidine nucleotides can produce CO₂, β-alanine and β-Aminoisobutyric acid, etc. Metabolic disorders of purine nucleotides and pyrimidine nucleotides can cause clinical symptoms, such as purine metabolism disorders, pyrimidine metabolism disorders. Therefore, it is important to analysis nucleoside/nucleotide and derivatives.

Our Nucleoside/Nucleotide Analysis Service

Historically, ATP concentration is measured with the established highly sensitive luciferin-luciferase luminescence assay technique appropriate for measuring localized ATP. Alternative analytical methods include HPLC-UV, LC-MS. While ATP is a useful biomarker for certain diseases, the significance of other nucleotides and nucleosides remains largely unexplored. Simultaneous quantification of ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP can't be achieved with the luciferin-luciferase assay. But don't worry, the Creative Proteomics team has successfully developed streamlined, rapid, and dependable LC-MS and HPLC-UV methodologies that we can identification and quantification more than 16 Nucleosides/Nucleotides.

Technological superiority

1. Professional detection and analysis capability: Experienced research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.

2. Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.

3. High veracity of data: For sample identification, qualitative and quantitative analysis of various nucleosides and nucleotides can be achieved efficiently and accurately, with low cost, high efficiency, and standard curve R²>0.99.

4. High resolution and sensitivity: AB SCIEX QTRAP 6500 Plus, AB SCIEX QTRAP 5500, et al.

Samples Requirement

• Normal Volume: 200 μL serum/plasma; 200 mg tissue, 2×10⁷ cells
• Minimal Volume: 50 μL serum/plasma; 50 mg tissue, 5×10⁶ cells.
• Any other samples such as body fluid, feces, cell culture medium supernatant.

Results Delivery

1. A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters, etc.

2. Raw data and data analysis results.

• Analytes are reported as μM or μg/mg (tissue), and variable-coefficient are generally<10%
• The name of the analytes, abbreviation, formula, molecular weight, and CAS# would also be included in the report.

How to place an order

At Creative Proteomics, many excellent and experienced experts will optimize the experimental protocol according to your requirement and guarantee the high-quality results for Nucleoside/Nucleotide Analysis Service. Creative Proteomics can provide a broad range of technologies for Nucleoside/Nucleotide Analysis Service. Please feel free to contact us by email to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.

NUDT22 promotes cancer growth through pyrimidine salvage

Journal: Oncogene

Published: 2023

Background

Cancer cells often face genetic instability due to oncogene-induced replication stress, which is partly due to inaccurate deoxynucleotide (dNTP) supply at replication forks. Traditional cancer treatments target dNTP synthesis pathways using anti-folates, thymidylate synthetase inhibitors, or ribonucleotide reductase (RNR) inhibitors. The role of the NUDIX family gene NUDT22, which encodes a UDP-glucose hydrolase converting UDP-glucose into uridine-monophosphate (UMP) and glucose-1-phosphate (G-1-P), has not been well explored in cancer research. NUDT22's biological function and its potential as a cancer vulnerability are the focus of recent studies.

Materials & Methods

Antibodies and Reagents:

• Antibodies: NUDT22 (Santa Cruz), β-Actin (Abcam), p53 (Santa Cruz), cMyc (Santa Cruz), γH2A.X (Millipore), RPA (Cell Signalling Technology), and others.
• Reagents: Drugs used include pyrazofurin, MPA, 6MP, brequinar, leflunomide, and others. SiRNA pools and plasmids for gene editing were sourced from Dharmacon, Addgene, and other providers.

Cell Culture:

Cell lines (U2OS, hTERT-RPE1, HA1EB, BJ-MYCER, HCT116, MCF7, MRC5-SV2, 16HBE14o) were cultured in respective media with supplements and antibiotics. Mycoplasma contamination was regularly checked, and cells were validated by STR profiling.

Gene Editing:

CRISPR/Cas9: Used for NUDT22 knockout with sgRNA sequences specific to each cell type. Cells were selected with puromycin, and knockout efficiency was confirmed by sequencing.

Dual-Luciferase Assay:

Transfected U2OS cells with NUDT22-luc2-pGL4.10 and CMV-hRluc-pGL4.75. Assayed luciferase activity using Hidex Sense.

Cell Transfection:

siRNA: Transfected using INTERFERin. Control siRNA was used for comparison.

Plasmid DNA: Transfected with jetPEI.

Drug Exposure:

Cells were treated with various drugs in a 384-well plate format. Viability was assessed using a resazurin assay after 4 days.

Western Blotting:

Performed using standard protocols with SDS gradient gels and a Trans-Blot Turbo system. Proteins were detected with LI-COR Odyssey FC or Bio-Rad Chemidoc MP.

RNA Extraction and qRT-PCR:

RNA was isolated using Direct-zol RNA Mini Prep or ReliaPrep kits. cDNA was synthesized, and gene expression was quantified using SYBR Green on Rotor-Gene Q or CFX96 machines.

ChIP-qPCR:

Performed on U2OS cells transfected with GFP or p53-IRES-GFP. Enrichment of P21 and NUDT22 promoter regions was measured.

DNA Fiber Assay:

Cells were pulse-labeled with CldU and IdU, and DNA fibers were visualized and measured using confocal microscopy.

EdU Incorporation:

Cells were treated with pyrazofurin, followed by EdU labeling and click reaction. Cells were imaged using ImageXpress XLS or Celldiscoverer7.

Statistical Analysis:

Unpaired two-tailed Student's t test or Mann–Whitney test for comparisons. Analyses were performed using GraphPad Prism.

Targeted Metabolomics for LC-MS Nucleotide Measurements:

Nucleotide concentrations were measured using UPLC-MRM/MS. Samples were processed and analyzed with a Waters Acquity UPLC system and Sciex QTRAP 6500 Plus MS.

In Vivo Studies:

MCF7-GFP-Luc cells were injected into NOD/SCID mice. Tumor growth was monitored with an IVIS Lumina II system using D-Luciferin.

Cell Fractionation:

Soluble and insoluble fractions were separated. The soluble fraction was lysed with Digitonin buffer, and the insoluble fraction with RIPA buffer.

Cell Cycle Analysis:

Cells were synchronized using a double thymidine block and analyzed at various time points post-release. CCNB1 antibody was used to confirm cell cycle phases.

Database Analysis:

Data from cBioPortal and UCSC Xenabrowser were analyzed for gene expression and overall survival related to NUDT22 alterations.

Results

NUDT22 Expression and Regulation

NUDT22 expression is notably upregulated in response to disruptions in glucose metabolism. This upregulation is associated with increased levels of uridine-monophosphate (UMP) and glucose-1-phosphate (G-1-P), which is part of a compensatory mechanism to manage altered glucose metabolism. The upregulation of NUDT22 is driven by the oncogene cMYC and is dependent on p53, which binds to the NUDT22 promoter, leading to its increased expression upon p53 stabilization.

Impact on Nucleotide Metabolism

NUDT22 plays a pivotal role in nucleotide metabolism, particularly in maintaining the balance of nucleotide pools necessary for DNA replication. In NUDT22 knockout (KO) cells, there is a significant reduction in deoxynucleotide triphosphate (dNTP) levels, leading to slower replication fork speeds. This effect is partially reversed by uridine supplementation, indicating NUDT22's crucial role in supplying nucleotides for DNA synthesis. Moreover, NUDT22 KO cells exhibit heightened sensitivity to pyrimidine synthesis inhibitors and ribonucleotide reductase (RNR) inhibitors, with a more pronounced effect observed in cancer cells compared to normal fibroblasts. This suggests that NUDT22 helps in managing nucleotide availability and responding to nucleotide metabolism disruptions.

Loss of NUDT22 potentiates inhibition of nucleotide metabolism.

Replication Stress and DNA Damage

The absence of NUDT22 leads to increased replication stress and DNA damage, as evidenced by higher levels of replication stress markers and DNA damage indicators. This stress is exacerbated by pyrimidine synthesis inhibitors but can be alleviated by uridine supplementation. Additionally, NUDT22 deficiency impairs cancer cell growth in both in vitro and xenograft models, underscoring its importance in cellular proliferation and replication stress management.

Reference

1. Walter, Melanie, et al. "NUDT22 promotes cancer growth through pyrimidine salvage." Oncogene 42.16 (2023): 1282-1293.