Aptamers as New Tools for Inhibiting Cytokine Activity Howard A. Young Laboratory of Experimental Immunology Cancer and Inflammation Program National Cancer Institute at Frederick Frederick, MD

Collaboration with: Nucleic Acid Synthetic Biology Resarch Team Systems and structural Biology Center Yokohama Institute, RIKEN Ichiro Hirao Team Leader RIKEN Center for Life Science Technologies President & CEO TagCyx Biotechnologies

Aptamers (from the Latin aptus - fit, and Greek meros - part) are oligonucleic acid or peptide molecules that bind to a specific target molecule.

Nat Biotechnol.Nat Biotechnol May;31(5): doi: /nbt Epub 2013 Apr 7. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Kimoto M Kimoto M 1, Yamashige R, Matsunaga K, Yokoyama S, Hirao I.Yamashige RMatsunaga KYokoyama SHirao I Abstract DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.

Genetic alphabet expansion of the central dogma by unnatural base pairs DNA aptamers, PCR detection RNA labeling, functional RNAs Unnatural proteins Next-generation biotechnology by synthetic xenobiology In vivo systems Ds Px Diagnostic and therapeutic applications Nat. Methods, 3, 729 (2006). Nucleic Acids Res., 37, e14 (2009). Nucleic Acids Res., 40, 2793 (2012).

DNA aptamer selection for protein targets Selection PCR amplification DNA aptamer Conventional DNA library Conventional aptamer selection Selection PCR amplification DNA aptamer New DNA library with five different bases Unnatural base New methods using unnatural base pair systems High affinity ? In vitro selection (SELEX) A.D. Ellington & J.W. Szostak, Nature, 346, 818 (1990). C. Tuerk & L. Gold, Science, 249, 505 (1990). D.L. Robertson & G.F. Joyce, Nature, 344, 467 (1990).

Systematic Evolution of Ligands by Exponential Enrichment SELEX involving the Ds−Px pair DsPx Selection PCR amplification DNA aptamer Target protein Nature Biotechnol., 31, (2013). 2 ） Increase in structural diversity because of no pairing partner of Ds because of no pairing partner of Ds １） Increase in hydrophobicity DNA aptamer Hydrophobic Ds Target protein Hydrophobic pocket

High affinity aptamer generation by an unnatural base pair system Selection PCRamplification Target protein Ds-aptamer : protein complex Ds-containing DNA library SELEX Ds-base New nucleic acid antibodies The binding abilities represented more than a 100-fold increase compared with those for conventional DNA aptamers. Nature Biotechnol., 31, (2013).

Anti-IFNγ DNA aptamer selection Ds T T C C 12 T A G G T T G G G G T G T A G G G C C GG A T C T A Ds T T T TA A 29 A Ds 40 G A ctcac Stem-1 Stem-2 GG >96% >99% <80% : DMS : DMS, protected by protein binding : KMnO 4 : KMnO 4, protected by protein binding : KMnO 4, enhanced by protein binding : MungBean nuclease, protected by protein binding Base conservation after the doped selection Chemical and enzymatic probing : MungBean nuclease Anti-IFNγ Ds x 3 (49-mer) Nature Biotechnol., 31, (2013).

Anti-IFNγ DNA aptamer selection Tm = 37.6 ˚C Tm = 33.1 ˚C Ds Anti-IFNγ Ds x 3 (49-mer) K D = nM 38 pM 38 pM Nature Biotechnol., 31, (2013) Response units (RU) Time (sec) IFNγ (nM) 1.25 A A A Anti-IFNγ A x 3 (49-mer) K D = 7.21 nM Time (sec) 600 Ramanathan, M. et al., Transplantation, 52, 612 (1994). Known anti-IFNγ DNA aptamer (26-mer) K D = 16.6 nM Time (sec) nM 20 nM 10 nM 5 nM 2.5 nM IFN-γ

Anti-IFNγ DNA aptamer selection Protein 20 nM Normalized Responses High selectivity of the anti-IFNγ aptamer (aptamer Ds x 3, 49-mer) VEGF 165 BSA VEGF 121 IFNγ EGF Thrombin Time (sec) Sensorgrams (SPR) for the binding of different proteins (20 nM) to 5′-biotinylated aptamer Ds x 3 (49-mer) in 1×PBS % NP40 Nature Biotechnol., 31, (2013).

Assay for Biological Activity Treat target cell (human breast tumor cell line) with IFN-  for 10 minutes Assay for phospho-STAT1 via flow cytometry

Aptamer effects on IFNγ induced pSTAT1 at 37˚C for 10 min 2 ng 10 ng 100 ng 38 pM IFNγ = 2 ng Aptameraddition Unpublished data 2 ng 10 ng 100 ng 16 nM Anal. Chem., 82, 1851 (2010).

Nuclease degradation and stabilization of aptamers 10 min 37˚CO/Nr.t.O/N37˚C 38 pM 10 min 37˚CO/Nr.t.O/N37˚C 5’ 3’ Unpublished data

Conclusions: 1. IFN-  aptamer interaction is extremely rapid 2. Aptamer blocks IFN-  interaction with cell surface receptor DNA-Aptamer Time Course and Effect of Wash out and Restimulation

Stabilization of DNA aptamers by attaching a mini-hairpin DNA sequence Ds (hr) In 96 ％ human serum Ds-DNA aptamer + Mini-hairpin DNA Unpublished data

Stability of the mini-hairpin DNAs against nucleases

Extraordinarily thermal stable mini-hairpin DNA (dGCGAAGC) Nucleic Acids Res., 17, 2223 (1989) and 22, 576 (1994). Tm = 76.5 ˚C

Aptamer (100 ng/ml) effects on IFNγ induced pSTAT1 at 37˚C, overnight Kd = 16 nM Unpublished data 200 ng/ml

Diagnostic and therapeutic applications of new DNA aptamers 20 Ds Unnatural-base DNA aptamers Mini-hairpin DNA High stable and high affinity DNA aptamers for imaging, diagnostics and therapeutics

Acknowledgements RIKEN Center for Life Science Technologies TagCyx Biotechnologies Dr. Ichiro Hirao NCI at Frederick Charlotte Hanson Michael Sanford