title: Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer.
abstract: DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.
authors.affiliation: Departments of Biochemistry and Physiology, Li Ka Shing Faculty of Medicine, and Faculty of Dentistry, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People's Republic of China.
authors.forename: Yee-Wai
authors.initials: YW
authors.lastname: Cheung
authors.forename: Jane
authors.initials: J
authors.lastname: Kwok
authors.forename: Alan W L
authors.initials: AW
authors.lastname: Law
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Masayo
authors.initials: M
authors.lastname: Kotaka
authors.forename: Julian A
authors.initials: JA
authors.lastname: Tanner
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D024202:Electrophoretic Mobility Shift Assay; D015723:Gene Library; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008288:Malaria / Q000175:diagnosis* / Q000201:enzymology; D008958:Models, Molecular; D009841:Oligonucleotides / Q000235:genetics; D010961:Plasmodium / Q000201:enzymology*; D011487:Protein Conformation; D052156:SELEX Aptamer Technique
chemical_list: D052157:Aptamers, Nucleotide; D015415:Biomarkers; D009841:Oligonucleotides; D007770:L-Lactate Dehydrogenase
keywords: SELEX; X-ray crystallography; malaria diagnosis; point-of-care diagnostics
references.citation: Nature. 2006 Nov 23;444 Suppl 1:39-48
references.pmid: 17159893
references.citation: J Biol Chem. 1999 Apr 9;274(15):10213-8
references.pmid: 10187806
references.citation: Anal Biochem. 2013 Aug 1;439(1):11-6
references.pmid: 23583275
references.citation: Biosens Bioelectron. 2012 May 15;35(1):291-296
references.pmid: 22459583
references.citation: Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):19971-6
references.pmid: 23139410
references.citation: Methods. 2000 Mar;20(3):310-8
references.pmid: 10694453
references.citation: Am J Trop Med Hyg. 2009 Apr;80(4):516-22
references.pmid: 19346368
references.citation: Structure. 2009 Nov 11;17(11):1476-84
references.pmid: 19913482
references.citation: Curr Pharm Des. 2012;18(14):2014-26
references.pmid: 22376117
references.citation: Nature. 1992 Feb 27;355(6363):850-2
references.pmid: 1538766
references.citation: Am J Clin Pathol. 2010 Oct;134(4):529-31
references.pmid: 20855632
references.citation: Nucleic Acids Res. 2012 Sep;40(16):8119-28
references.pmid: 22669903
references.citation: Faraday Discuss. 2011;149:49-61; discussion 63-77
references.pmid: 21413174
references.citation: Clin Infect Dis. 2012 Jun;54(11):1637-41
references.pmid: 22550113
references.citation: Nucleic Acids Res. 2011 Sep 1;39(17):7858-67
references.pmid: 21715374
references.citation: Nat Struct Biol. 1996 Nov;3(11):912-5
references.pmid: 8901865
references.citation: Annu Rev Anal Chem (Palo Alto Calif). 2009;2:241-64
references.pmid: 20636061
references.citation: Int J Mol Sci. 2012;13(8):10537-10552
references.pmid: 22949878
references.citation: J Biol Chem. 2004 Jul 23;279(30):31429-39
references.pmid: 15117937
references.citation: Angew Chem Int Ed Engl. 2006 Dec 4;45(47):7955-9
references.pmid: 17094149
references.citation: Curr Opin Pharmacol. 2010 Oct;10(5):557-62
references.pmid: 20638902
references.citation: Science. 1990 Aug 3;249(4968):505-10
references.pmid: 2200121
references.citation: Structure. 2000 May 15;8(5):R105-10
references.pmid: 10801499
references.citation: Angew Chem Int Ed Engl. 2009;48(15):2672-89
references.pmid: 19319884
references.citation: Angew Chem Int Ed Engl. 2012 Jul 9;51(28):6925-8
references.pmid: 22639438
references.citation: Acc Chem Res. 2011 Dec 20;44(12):1349-58
references.pmid: 21819040
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: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Curr Protoc Nucleic Acid Chem. 2002 Aug;Chapter 12:Unit 12.2
references.pmid: 18428889
references.citation: Methods Enzymol. 2003;374:300-21
references.pmid: 14696379
references.citation: Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):32-41
references.pmid: 17164524
references.citation: Blood. 1993 Jun 15;81(12):3271-6
references.pmid: 8507864
references.citation: Angew Chem Int Ed Engl. 2005 Dec 16;45(1):90-4
references.pmid: 16292781
references.citation: Angew Chem Int Ed Engl. 2010 Feb 1;49(6):1052-6
references.pmid: 20084650
references.citation: Exp Parasitol. 2008 Oct;120(2):135-41
references.pmid: 18619439
references.citation: J Biol Chem. 1993 Aug 25;268(24):17651-4
references.pmid: 8102368
references.citation: Am J Trop Med Hyg. 2006 May;74(5):750-4
references.pmid: 16687674