Ichiro Hirao is a team leader and principal research scientist of Institute of Bioengineering and Nanotechnology (IBN), A*STAR in Singapore. He obtained his Ph.D. from Tokyo Institute of Technology in 1983. In 1984, he worked at The University of Tokyo, as a research associate. In 1992, he became an associate professor at Tokyo University of Pharmacy and Life Sciences. In 1995, he moved to Dr. Andrew Ellington’s laboratory at Indiana University. In 1997, to start the unnatural base pair studies, he returned to Japan and joined JST, as a group leader. In 2002, he continued his work as a professor at The University of Tokyo. From 2006 to 2015, he had been managing a synthetic biology team in RIKEN. In 2007, he founded the venture company ‘TagCyx Biotechnologies’.
Creation of high-affinity DNA aptamers using a genetic alphabet expansion system
DNA aptamers could be an alternative of protein-based antibodies for diagnostics and therapeutics, if they can be routinely generated as a form with increased affinity and selectivity to targets and high stability against nucleases. In contrast to 20 variable amino acids of proteins, only four different nucleotides with A, G, C, and T seriously restrict the DNA aptamer functionality. Thus, genetic alphabet expansion technology using an artificial base pair (unnatural base pair) might improve the affinity of DNA aptamers by introducing unnatural bases as an additional extra base with different chemical and physical properties. Since general SELEX methods are performed by repetition cycles of selection and PCR amplification to generate DNA aptamers, thus DNA libraries containing unnatural bases must be amplified by PCR.
We developed an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) that functions as a third base pair with high fidelity in replication (Nucleic Acids Res., 40, 2793 (2012)., Nucleic Acids Res., 37, e14 (2009)., Nat. Methods, 3, 729 (2006).), and applied it to SELEX involving PCR amplification using the Ds–Px pair (Nat. Biotechnol., 31, 453-457 (2013).). We added the highly hydrophobic Ds bases as a fifth base into a random region of DNA libraries and performed the SELEX procedure using the Ds-containing DNA library. Then, we generated high-affinity unnatural-base DNA aptamers targeting VEGF165 (Kd = ~1 pM), interferon-γ (Kd = 38 pM), and vWF (Kd = 75 pM). Only few Ds bases in the aptamers significantly augmented their affinity.
Recently, we also succeeded in the stabilization of these unnatural-base DNA aptamers against nucleases by introducing a mini-hairpin DNA sequence (Sci. Rep., 5, 18478 (2015).). Twenty-five years ago, I accidentally found a series of short DNA fragments with GCGAAAGC or GCGAAGC, which form a compact hairpin structure with two G–C and one non-canonical (shared) G–A pairs and exhibit high thermal stability with more than 75°C of the melting temperature. These mini-hairpin DNA fragments also have the high resistance to nuclease digestion. Thus, we added the mini-hairpin sequence at the 3′-terminus of DNA aptamers (to resist exonucleases) and replaced hairpin structures in DNA aptamers with the mini-hairpin structure (to resist endonucleases). We also replaced the A–T pairs in the stem regions of DNA aptamers with G–C pairs. By this stabilization method, more than 70% of the anti-interferon-γ DNA aptamer survived in a human serum at 37°C after 3 days.
Here, I will introduce the high-affinity DNA aptamer generation by the genetic alphabet expansion and the stabilization using the mini-hairpin DNA. Biological activities using cultured cells and the generation of DNA aptamers that specifically bind to breast cancer cell lines by Cell-SELEX involving the genetic alphabet expansion will also be discussed.
Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications. K. Matsunaga, M. Kimoto, C. Hanson, M. Sanford, H.A. Young, I. Hirao, Sci. Rep., 5, 18478 (2015).
Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry. T. Someya, A. Ando, M. Kimoto, I. Hirao, Nucleic Acids Res.,43, 6665-6676 (2015).
Generation of high-affinity DNA aptamers using an expanded genetic alphabet. M. Kimoto, R. Yamashige, K. Matsunaga, S. Yokoyama, I. Hirao, Nat. Biotechnol., 31, 453-457 (2013).
Highly specific unnatural base pair systems as a third base pair for PCR amplification. R. Yamashige, M. Kimoto, Y. Takezawa, A. Sato, T. Mitsui, S. Yokoyama, I. Hirao, Nucleic Acids Res., 40, 2793-2806 (2012).