title: Generation of high-affinity DNA aptamers using an expanded genetic alphabet.
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 0.038 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.
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama, Kanagawa, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Rie
authors.initials: R
authors.lastname: Yamashige
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Matsunaga
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D052157:Aptamers, Nucleotide / Q000138:chemical synthesis* / Q000235:genetics*; D001665:Binding Sites; D015723:Gene Library
chemical_list: D052157:Aptamers, Nucleotide
references.citation: Biopolymers. 2009 Feb;91(2):145-56
references.pmid: 19025993
references.citation: Curr Opin Pharmacol. 2010 Oct;10(5):557-62
references.pmid: 20638902
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):12005-10
references.pmid: 22773812
references.citation: Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(7):345-67
references.pmid: 22850726
references.citation: Transpl Immunol. 1994 Dec;2(4):285-92
references.pmid: 7704538
references.citation: Science. 1990 Aug 3;249(4968):505-10
references.pmid: 2200121
references.citation: Curr Opin Drug Discov Devel. 2003 Mar;6(2):253-61
references.pmid: 12669461
references.citation: J Biol Chem. 1998 Aug 7;273(32):20556-67
references.pmid: 9685413
references.citation: Nat Methods. 2006 Sep;3(9):729-35
references.pmid: 16929319
references.citation: Nat Rev Drug Discov. 2006 Feb;5(2):123-32
references.pmid: 16518379
references.citation: Expert Opin Ther Pat. 2009 Nov;19(11):1603-13
references.pmid: 19852719
references.citation: Anal Chem. 2010 Mar 1;82(5):1851-7
references.pmid: 20121141
references.citation: J Am Chem Soc. 2011 Sep 28;133(38):15105-12
references.pmid: 21842904
references.citation: J Am Chem Soc. 2011 Dec 14;133(49):19878-88
references.pmid: 21981600
references.citation: Nucleic Acids Res. 2009 Feb;37(2):e14
references.pmid: 19073696
references.citation: Nucleic Acids Res. 1995 Oct 25;23(20):4220-1
references.pmid: 7479087
references.citation: Biochemistry. 2004 Mar 23;43(11):3214-21
references.pmid: 15023071
references.citation: Nat Chem. 2011 Jul 24;3(9):697-703
references.pmid: 21860458
references.citation: Nucleic Acids Res. 2012 Mar;40(6):2793-806
references.pmid: 22121213
references.citation: PLoS One. 2010 Dec 07;5(12):e15004
references.pmid: 21165148
references.citation: Nat Rev Drug Discov. 2010 Jul;9(7):537-50
references.pmid: 20592747
references.citation: Nature. 1990 Aug 30;346(6287):818-22
references.pmid: 1697402
references.citation: Nucleic Acids Res. 2007;35(13):4238-49
references.pmid: 17576683
references.citation: Science. 2012 Apr 20;336(6079):341-4
references.pmid: 22517858
references.citation: Anal Chem. 2010 Oct 1;82(19):8131-6
references.pmid: 20815336
references.citation: Chem Soc Rev. 2011 Dec;40(12):5680-9
references.pmid: 21556437