1. A proposal for: High affinity RNA aptamers as antagonists for AT 2 receptors Tina Stutzman Nick Swenson 20.109 May 12, 2010

2. High affinity RNA aptamers as an antagonist for AT 2 receptors

3. Hypertension is related to renal and cardiac failure Hypertension (high blood pressure) Treated by: Angiotensin Converting Enzyme (ACE) Inhibitors Angiotensin II Type I (AT 1 ) receptor (ARB) blocker Side effects include coughing (12%) and angioedema (inflammation) Untreated leads to: Kidney Disease Cardiac Disease Stroke

4. Renin-Angiotensin System (RAS) involved in regulation of blood pressure Angiotensin I Angiotensin II Liver Renin ACE Angiotensin II Type I Receptor (AT 1 ) Increased Blood Pressure Angiotensin II Type 2 Receptor (AT 2 ) Angiotenisinogen Angiotensin Converting Enzyme (ACE) Inhibitors Angiotensin II Type I (AT 1 ) receptor (ARB) blocker

5. ARB drugs inhibit AT 1 receptors and decrease blood pressure Angiotensin II Angiotensin II Type I (AT 1 ) Receptor ARB Increased Blood Pressure Angiotensin II Type 2 Receptor (AT 2 ) Angiotensin II type I (AT 1 ) receptor blocking (ARB) drugs prevent AngII from activating vascular smooth muscle cells to constrict Current ARB drugs include losartan and valsartan – Mimic the structure of angiotensin II to bind to active site of receptor Angiotensin II bound to AT 2 increases with bound ARB Bradykinin Nitric Oxide

6. ARB drugs inhibit AT 1 receptors and decrease blood pressure Angiotensin II Angiotensin II Type I (AT 1 ) Receptor ARB Angiotensin II type I (AT 1 ) receptor blocking (ARB) drugs prevent AngII from activating vascular smooth muscle cells to constrict Current ARB drugs include losartan and valsartan – Mimic the structure of angiotensin II to bind to active site of receptor Angiotensin II bound to AT 2 increases with bound ARB Angiotensin II Type 2 Receptor (AT 2 ) Decrease d Blood Pressure Cough, Angioedema Bradykinin Nitric Oxide Bradykinin

7. Aptamer bound to AT 2 may decrease the production of bradykinin RNA aptamer selected for the AT 2 receptor will inhibit activation function Bradykinin production cascade will not be activated Frequency of cough and angioedema could decrease with little affect on the blood pressure regulation Angiotensin II Type 2 Receptor (AT 2 ) Bradykinin Cough, Angioedema Angiotensin II

8. Experiment overview Isolating and immobilize AT 2 receptor Isolating and immobilize AT 2 receptor Use SELEX to select aptamers that bind AT 2 receptor Assay effect on bradykinin production with ELISA Evaluate Kd with dot-blot analysis

9. Express AT 2 Receptors Tag intracellular domain with an antibody binding site Solubilize AT 2 in a micelle Immobilize on glass beads AT 2 Receptor Immobilization R. Carey et al, Hypertension 35, 155-163 (2000) AT2 receptor structure

10. Selection and evaluation of binding aptamers Use 11 rounds of SELEX to select binding RNA aptamers RT-PCR to cDNA RT-PCR to cDNA Clone into vector Sequence Mfold analysis for structure Isolate unique aptamers Expected Results: Aptamers A1, A2, A3 Variable region = 40 nucleotides Negative selection on bead, strepavidin, BSA- biotin, antibody

11. Dot-blot analysis to determine aptamer-AT 2 K d A1 A2 A3 10 25 50 75 100 250 500 AT 2 (ng) Y. Jian et al. Oncogene 28, 4201–4211 (2009) Expected Results: Aptamers A1 and A2 show stronger binding to AT 2 than aptamer A3

12. Bradykinin production assay Sandwich ELISA to evaluate bradykinin levels in vascular smooth muscle cells in vitro incubated with known Angiotensin II concentrations ARB ARB + A2 ARB + A1 Measure Bradykinin Expected: No ARB Control Baseline Bradykinin levels Increased Bradykinin levels Baseline Bradykinin levels Increased Bradykinin levels

13. Expected bradykinin ELISA results High affinity aptamer A1 reduces bradykinin production in the presence of ARB Bradykinin (pg) 1000 1500 2000 Control No ARB ARB ARB + A1ARB + A2

14. Necessary Resources

15. Societal Impact