Aptabody-aptatope interactions in aptablotting assays.

We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein-aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody-aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.

[1]  P. Labute,et al.  Assessment of fully automated antibody homology modeling protocols in molecular operating environment , 2014, Proteins.

[2]  G. Węgrzyn,et al.  A novel method for screening the glutathione transferase inhibitors , 2009, BMC Biochemistry.

[3]  Hua Shi,et al.  RNA aptamers that functionally interact with green fluorescent protein and its derivatives , 2011, Nucleic acids research.

[4]  L. Maher,et al.  RNA aptamer inhibitors of a restriction endonuclease , 2015, Nucleic acids research.

[5]  R. Colman,et al.  Heterodimers of wild‐type and subunit interface mutant enzymes of glutathione S‐transferase A1–1: Interactive or independent active sites? , 2004, Protein science : a publication of the Protein Society.

[6]  G. Sun,et al.  A modified pGEX expression system that eliminates degradation products and thrombin from the recombinant protein. , 1995, Analytical biochemistry.

[7]  Ji-Young Ahn,et al.  Defining the copper binding aptamotif and aptamer integrated recovery platform (AIRP). , 2017, Nanoscale.

[8]  Yang-Hoon Kim,et al.  Preventing the formation of positive transcription elongation factor b by human cyclin T1-binding RNA aptamer for anti-HIV transcription , 2012, AIDS.

[9]  Dieter Braun,et al.  Protein-binding assays in biological liquids using microscale thermophoresis. , 2010, Nature communications.

[10]  M. Komiyama,et al.  Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy , 2011, Nature communications.

[11]  S. Jayasena.  Aptamers: an emerging class of molecules that rival antibodies in diagnostics. , 1999, Clinical chemistry.

[12]  Xingyu Jiang,et al.  Nanomaterials for Ultrasensitive Protein Detection , 2013, Advanced materials.

[13]  H. Gaub,et al.  Dynamic force spectroscopy of the digoxigenin–antibody complex , 2006, FEBS letters.

[14]  Y. Chai,et al.  Supersandwich-type electrochemiluminescenct aptasensor based on Ru(phen)3(2+) functionalized hollow gold nanoparticles as signal-amplifying tags. , 2013, Biosensors & bioelectronics.

[15]  L. Gervais,et al.  Microfluidic Chips for Point‐of‐Care Immunodiagnostics , 2011, Advanced materials.

[16]  V. Stecher,et al.  Single-step purification of rat C-reactive protein and generation of monospecific C-reactive protein antibody. , 1988, Journal of Chromatography A.

[17]  C. Kessler,et al.  Non-radioactive labeling and detection of nucleic acids. II. Optimization of the digoxigenin system. , 1990, Biological chemistry Hoppe-Seyler.

[18]  P. Chames,et al.  Therapeutic Antibodies for the Treatment of Pancreatic Cancer , 2010, TheScientificWorldJournal.

[19]  H. Smidt,et al.  Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches , 2012, International journal of molecular sciences.

[20]  B. Sullenger,et al.  Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker. , 2012, The Journal of clinical investigation.

[21]  Ji-Young Ahn,et al.  Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes. , 2015, Biosensors & bioelectronics.

[22]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[23]  J. Rodrigues,et al.  Integrative computational modeling of protein interactions , 2014, The FEBS journal.

[24]  Anthony D. Keefe,et al.  Aptamers as therapeutics , 2010, Nature Reviews Drug Discovery.

[25]  C. Kessler,et al.  Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. , 1991, Analytical biochemistry.

[26]  C. Evelo,et al.  Inhibition of human glutathione S-transferase P1-1 by tocopherols and alpha-tocopherol derivatives. , 2001, Biochimica et biophysica acta.

[27]  Burnette Wn.  Western blotting : remembrance of past things. , 2009 .

[28]  P. Jordan,et al.  PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences , 2001, Molecular pathology : MP.

[29]  Ji-Young Ahn,et al.  Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC) , 2015, Scientific Reports.

[30]  Leonardo L. G. Ferreira,et al.  Molecular Docking and Structure-Based Drug Design Strategies , 2015, Molecules.

[31]  Janusz M. Bujnicki,et al.  NPDock: a web server for protein–nucleic acid docking , 2015, Nucleic Acids Res..

[32]  Benjamin Williams-Hubbard,et al.  DommiMOE: An implementation of ligand field molecular mechanics in the molecular operating environment , 2005, J. Comput. Chem..