1. Samuel Kirkeng 7 December, 2017
Nucleic Acid Programmable Protein Array (NAPPA) as a tool for diagnostics
Samuel Kirkeng
7 December, 2017

2. What makes NAPPA special?
Cell-free expression
Lysate washed over slides provides material for coupled transcription/translation
High-throughput
Thousands of DNA sequences coding for proteins can be spotted simultaneously
Less time consuming
Purify only the DNA, instead of expressing then purifying the proteins themselves
Printing DNA is more reliable
Greater accuracy and

3. Basic Microarray Methodology
Proteins marked with fluorescence
Slide or chip is arrayed with different wells/washed with a coat that will immobilize, hold, protect, and orient proteins
Epoxy or other material
Expose binding sites, prevent the protein from denaturing
Capture molecules
antibodies
Probe molecules
Fluorescently labelled proteins
Interactions between labeled proteins and immobilized protein results in fluorescence.
Laser scanner analyzes the fluorescence

4. NAPPA methodology Slide coats Spot plasmid DNA Express proteins
Aminosilane binds phosphate groups in DNA to hold
them in place
Spot plasmid DNA
Immobilize DNA and capture antibody
Bovine serum albumin to increase binding
Anti-GST to provide amino acid groups as linkers
Express proteins
Transcription/Translation system
Incubation/Washing
30C and 15C and then washed with PBST (phosphate buffered saline)
Optimization and Analysis

5. Case study: NAPPA in ovarian cancer research
Identify the Tumor-associated autoantibodies (TAAb) biomarkers that could be used to tell cancerous patients from those that were healthy or had a benign form of the cancer
TAAb= autoantibodies generated due to overexpression/mutation of proteins
A few TAAbs and antigens against TAAbs have been identified in patients, but identifying more could lead to faster and more accurate diagnosis
735 antigen sites (to select against the TAAb markers), analysis of over 10,000 human proteins between 30 healthy individuals, 30 individuals with benign ovarian cancer, and 30 with ovarian cancer

6. Results Largest screen with a protein array done on ovarian cancer
Identified several new biomarkers
NUDT proteins (some of which may be involved in other cancers)
p53, CTAG2 may indicate particularly strong forms of ovarian cancer
TRIM39
PVR

7. Case study: NAPPA in HPV-related cancer research (cervical cancer)
600,000 cancers are related to Human Pampillomavirus each year
The virus itself has a small double-stranded sequence, but there are over 100 forms of HPV
In response to HPV cancers, an HPV-specific IgG antibody is produced

8. Results 98 genes were spotted from 12 different strains of HPV
Identified
HPV 16 E1, E2
HPV 31 E2
HPV 52 E7

10. Katchman, B. A. , Chomwell, D. , Wallstrom, G. , Vitonis, A. F
Katchman, B. A., Chomwell, D., Wallstrom, G., Vitonis, A. F., LaBaer, J., Cramer, D. W., & Anderson, K. S. (July 2017). Autoantibody biomarkers for the detection of serous ovarian cancer. Gynecologic Oncology, 146(1), Retrieved November 28, 2017, from ?via%3Dihub
Diez, P., Gonzalez-Gonzalez, M., Lourido, L., Degano, R. M., Ibarrola, N., Casado- Vela, J., Fuentes, M. (2015). NAPPA as a Real New Method for Protein Microarray Generation. Microarrays, 4(2), Retrieved December 1, 2017, from
Ewaisha, R., Meshay, I., Resnik, J., Katchman, B. A. and Anderson, K. S. (2016), Programmable protein arrays for immunoprofiling HPV-associated cancers. Proteomics, 16: 1215– doi: /pmic , from