Generation of High-affinity DNA Aptamers Using an Expanded Genetic Alphabet

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2013 Apr 9

PMID 23563318

Citations 143

Authors

Michiko Kimoto

Rie Yamashige

Ken-Ichiro Matsunaga

Shigeyuki Yokoyama

Ichiro Hirao

Affiliations

Soon will be listed here.

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.

Citing Articles

L-Histidine Modulates the Catalytic Activity and Conformational Changes of the HD3 Deoxyribozyme.

Sakimoto N, Imanaka H, Tomita-Sudo E, Akita T, Kawakami J Genes (Basel). 2024; 15(11).

PMID: 39596681 PMC: 11594175. DOI: 10.3390/genes15111481.

Recent advances in aptamer discovery, modification and improving performance.

Fallah A, Fooladi A, Havaei S, Mahboobi M, Sedighian H Biochem Biophys Rep. 2024; 40:101852.

PMID: 39525567 PMC: 11546948. DOI: 10.1016/j.bbrep.2024.101852.

Plug-and-play protein biosensors using aptamer-regulated in vitro transcription.

Lee H, Xie T, Kang B, Yu X, Schaffter S, Schulman R Nat Commun. 2024; 15(1):7973.

PMID: 39266511 PMC: 11393120. DOI: 10.1038/s41467-024-51907-4.

Recognition and Sequencing of Mutagenic DNA Adduct at Single-Base Resolution Through Unnatural Base Pair.

Wang H, Tie W, Zhu W, Wang S, Zhang R, Duan J Adv Sci (Weinh). 2024; 11(40):e2404622.

PMID: 39225557 PMC: 11515917. DOI: 10.1002/advs.202404622.

Reverse transcription as key step in RNA evolution with unnatural base pairs.

Hoffmann E, De Pascali M, Neu L, Domnick C, Solda A, Kath-Schorr S RSC Chem Biol. 2024; 5(6):556-566.

PMID: 38846072 PMC: 11151862. DOI: 10.1039/d4cb00084f.

References

Potty A, Kourentzi K, Fang H, Jackson G, Zhang X, Legge G . Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor. Biopolymers. 2008; 91(2):145-56. DOI: 10.1002/bip.21097. View

Bunka D, Platonova O, Stockley P . Development of aptamer therapeutics. Curr Opin Pharmacol. 2010; 10(5):557-62. DOI: 10.1016/j.coph.2010.06.009. View

Malyshev D, Dhami K, Quach H, Lavergne T, Ordoukhanian P, Torkamani A . Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc Natl Acad Sci U S A. 2012; 109(30):12005-10. PMC: 3409741. DOI: 10.1073/pnas.1205176109. View

Hirao I, Kimoto M . Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma. Proc Jpn Acad Ser B Phys Biol Sci. 2012; 88(7):345-67. PMC: 3422687. DOI: 10.2183/pjab.88.345. View

Tam S, Huey B, Li Y, Lui G, Hwang D, Lantz M . Suppression of interferon-gamma induction of MHC class II and ICAM-1 by a 26-base oligonucleotide composed of deoxyguanosine and deoxythymidine. Transpl Immunol. 1994; 2(4):285-92. DOI: 10.1016/0966-3274(94)90004-3. View

Tuerk C, Gold L . Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990; 249(4968):505-10. DOI: 10.1126/science.2200121. View

Vater A, Klussmann S . Toward third-generation aptamers: Spiegelmers and their therapeutic prospects. Curr Opin Drug Discov Devel. 2003; 6(2):253-61. View

Ruckman J, Green L, Beeson J, Waugh S, Gillette W, Henninger D . 2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem. 1998; 273(32):20556-67. DOI: 10.1074/jbc.273.32.20556. View

Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A . An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nat Methods. 2006; 3(9):729-35. DOI: 10.1038/nmeth915. View

10.

Ng E, Shima D, Calias P, Cunningham Jr E, Guyer D, Adamis A . Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006; 5(2):123-32. DOI: 10.1038/nrd1955. View

11.

Majumder P, Gomes K, Ulrich H . Aptamers: from bench side research towards patented molecules with therapeutic applications. Expert Opin Ther Pat. 2009; 19(11):1603-13. DOI: 10.1517/13543770903313746. View

12.

Tuleuova N, Jones C, Yan J, Ramanculov E, Yokobayashi Y, Revzin A . Development of an aptamer beacon for detection of interferon-gamma. Anal Chem. 2010; 82(5):1851-7. DOI: 10.1021/ac9025237. View

13.

Yang Z, Chen F, Alvarado J, Benner S . Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J Am Chem Soc. 2011; 133(38):15105-12. PMC: 3427765. DOI: 10.1021/ja204910n. View

14.

Seo Y, Malyshev D, Lavergne T, Ordoukhanian P, Romesberg F . Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs. J Am Chem Soc. 2011; 133(49):19878-88. PMC: 3988912. DOI: 10.1021/ja207907d. View

15.

Kimoto M, Kawai R, Mitsui T, Yokoyama S, Hirao I . An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Res. 2008; 37(2):e14. PMC: 2632903. DOI: 10.1093/nar/gkn956. View

16.

Williams K, Bartel D . PCR product with strands of unequal length. Nucleic Acids Res. 1995; 23(20):4220-1. PMC: 307365. DOI: 10.1093/nar/23.20.4220. View

17.

Hirao I, Harada Y, Nojima T, Osawa Y, Masaki H, Yokoyama S . In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA. Biochemistry. 2004; 43(11):3214-21. DOI: 10.1021/bi0356146. View

18.

Xiang Y, Lu Y . Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nat Chem. 2011; 3(9):697-703. PMC: 3299819. DOI: 10.1038/nchem.1092. View

19.

Yamashige R, Kimoto M, Takezawa Y, Sato A, Mitsui T, Yokoyama S . Highly specific unnatural base pair systems as a third base pair for PCR amplification. Nucleic Acids Res. 2011; 40(6):2793-806. PMC: 3315302. DOI: 10.1093/nar/gkr1068. View

20.

Gold L, Ayers D, Bertino J, Bock C, Bock A, Brody E . Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One. 2010; 5(12):e15004. PMC: 3000457. DOI: 10.1371/journal.pone.0015004. View