Rainier Ababao's ONP aptamer project (2014)
RNA aptamer selection against o-nitrophenol to assemble a biomarker for oxidative stress
Approximately 600,000 deaths in the United States can be attributed to heart disease every year1, and there is a strong biochemical link between oxidative stress and atherosclerosis4. Although it is not yet completely sure whether oxidative stress is a direct cause or marker of the disease3, the mere presence of it indicates a huge health risk. In periods of oxidative stress, excess nitric oxide and superoxide intermediates will posttranslationally nitrosylate tyrosine into nitrotyrosine. Nitrotyrosine contains a small molecule called o-nitrophenol that uniquely identifies it from other molecules in cellular pathways (Fig. 1).
Figure 1: Chemical structures of nitrotyrosine and o-nitrophenol. We avoid selecting against pure nitrotyrosine because of the possibility that RNA species will bind to the amino or carboxyl groups common to all amino acids, defeating the purpose of an aptamer.
A better tool for detecting nitrotyrosine could generate quicker diagnoses for cardiovascular disease. An aptamer, which is an oligonucleotide that binds to a specific target molecule6, could be used as an oxidative stress biomarker and point-of-care diagnostic for cardiovascular disease. A technique used to produce aptamers is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX)2. This particular in vitro selection begins with a synthesized pool containing 1013 unique RNA species. Small his-tagged peptides containing nitrotyrosine are immobilized onto beads and incubated with the pool to allow RNA species with some affinity for the target to bind, the species that bind are eluted, amplified exponentially, and prepared for another round to be incubated with target-immobilized beads again.
Several rounds of in vitro RNA selection from the N71 pool will be performed on o-nitrophenol. The stringency of the selection will be increased each round, predominantly by increasing the number of washes, the RNA-to-target concentration ratio, and introducing competitive elutions. This will increase number of oligos competing for binding sites so the tightest binders with slowest off-rate kinetics remain, thus ensuring that species with the highest affinity for the target will be selected for.
The target is a his-tagged peptide containing nitrotyrosine, synthesized with materials from the Ellington lab, at no virtually no cost to the FRI. The target’s molecular weight is 139.1088 Da but is bound to a peptide with molecular weight 2417.3455 Da. The RNA will be partitioned using Dynabeads® His-Tag Isolation and Pulldown by Life Technologies (10103D), which can be purchased at https://www.lifetechnologies.com/order/catalog/product/10103D (800-955-6288) in 2 mL aliquots for $197.00. Most negative selections will be performed against either a his-tagged peptide containing tyrosine in place of nitrotyrosine, which was developed at no cost to the FRI, or the beads described above.
1. Murphy SL, Xu JQ, Kochanek KD. “Deaths: Final data for 2010.” Natl Vital Stat Rep. 2013;61(4).
2. Ellington, A.D., Szostak, J.W. (1990) “In vitro selection of RNA molecules that bind specific ligands”. Nature (346): 818-822.
3. Daiber, A., Münzel, T. (2012) “Increased Circulating Levels of 3-Nitrotyrosine Autoantibodies: Marker or Maker of Cardiovascular Disease?”. Circulation Amer. Heart Assoc. 13;126(20):2371-3
4. Harrison D., Griendling K.K., Landmesser U., Hornig B., Drexler H., (2003) “Role of oxidative stress in atherosclerosis”, The American Journal of Cardiology, Volume 91, Issue 3, Supplement, 6 February 2003: 7-11, ISSN 0002-9149.