{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T11:27:20Z","timestamp":1779881240816,"version":"3.53.1"},"reference-count":50,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2014,5,26]],"date-time":"2014-05-26T00:00:00Z","timestamp":1401062400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide\u2013receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'\u21925' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense\u2013antisense (epitope\u2013paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 \u00b5M, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used  for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.<\/jats:p>","DOI":"10.3390\/ijms15069209","type":"journal-article","created":{"date-parts":[[2014,5,27]],"date-time":"2014-05-27T02:36:58Z","timestamp":1401158218000},"page":"9209-9223","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin"],"prefix":"10.3390","volume":"15","author":[{"given":"Nikola","family":"\u0160tambuk","sequence":"first","affiliation":[{"name":"Center for Nuclear Magnetic Resonance, Ru\u0111er Bo\u0161kovi\u0107 Institute, Bijeni\u010dka cesta 54,  10002 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoran","family":"Manojlovi\u0107","sequence":"additional","affiliation":[{"name":"Croatian Institute for Toxicology and Antidoping, Borongajska 83 g,  10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2621-8257","authenticated-orcid":false,"given":"Petra","family":"Tur\u010di\u0107","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Domagojeva 2, 10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roko","family":"Martini\u0107","sequence":"additional","affiliation":[{"name":"Department for Clinical Pathophysiology, Clinical Hospital Centre Split, \u0160oltanska 1,  21000 Split, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pa\u0161ko","family":"Konjevoda","sequence":"additional","affiliation":[{"name":"Center for Nuclear Magnetic Resonance, Ru\u0111er Bo\u0161kovi\u0107 Institute, Bijeni\u010dka cesta 54,  10002 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tin","family":"Weitner","sequence":"additional","affiliation":[{"name":"Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kova\u010di\u0107a 1, 10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piotr","family":"Wardega","sequence":"additional","affiliation":[{"name":"NanoTemper Technologies GmbH, Fl\u00f6\u00dfergasse 4, 81369 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Gabri\u010devi\u0107","sequence":"additional","affiliation":[{"name":"Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kova\u010di\u0107a 1, 10000 Zagreb, Croatia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2014,5,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1002\/1439-7633(20020301)3:2\/3<136::AID-CBIC136>3.0.CO;2-7","article-title":"Specific interactions between sense and complementary peptides: The basis for the proteomic code","volume":"3","author":"Heal","year":"2002","journal-title":"ChemBioChem"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1742-4682-4-45","article-title":"The proteonomic code: A molecular recognition code for proteins","volume":"4","author":"Biro","year":"2007","journal-title":"Theor. Biol. Med. Model."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1038\/nm0995-876","article-title":"Genetic origin of protein shape and interaction rules","volume":"1","author":"Blalock","year":"1995","journal-title":"Nat. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"7331","DOI":"10.3390\/molecules16097331","article-title":"Interaction of \u03b1-melanocortin and its pentapeptide antisense LVKAT: Effects on hepatoprotection in male CBA mice","volume":"16","author":"Houra","year":"2011","journal-title":"Molecules"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/j.it.2005.05.001","article-title":"Role of complementary proteins in autoimmunity: An old idea re-emerges with new twists","volume":"26","author":"McGuire","year":"2005","journal-title":"Trends Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.thbio.2005.02.001","article-title":"Molecular recognition theory of the complementary (antisense) peptide interactions","volume":"123","author":"tambuk","year":"2005","journal-title":"Theory Biosci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1002\/jmr.690","article-title":"Peptide self-aggregation and peptide complementarity as bases for the evolution of peptide receptors: A review","volume":"18","year":"2005","journal-title":"J. Mol. Recognit."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Elliott, S.G., Foote, M.A., and Molineux, G. (2009). Erythropoietins, Erythropoietic Factors and Erythropoiesis, Birkh\u00e4user Verlag.","DOI":"10.1007\/978-3-7643-8698-6"},{"key":"ref_9","unstructured":"Walsh, G. (2003). Biopharmaceuticals: Biochemistry and Biotechnology, Wiley. [2nd ed.]."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Thieme, D., and Hemmersbach, P. (2010). Doping in Sports, Springer-Verlag.","DOI":"10.1007\/978-3-540-79088-4"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1182\/blood.V77.6.1203.1203","article-title":"Evidence for the location of the receptor-binding site of human erythropoietin at the carboxyl-terminal domain","volume":"77","author":"Fibi","year":"1991","journal-title":"Blood"},{"key":"ref_12","unstructured":"Brown, F., Doughan, G., Hoey, E.M., Martin, S.J., Rima, B.K., and Trudgett, A. (1993). Vaccine Design, Wiley."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3824","DOI":"10.1073\/pnas.78.6.3824","article-title":"Prediction of protein antigenic determinants from amino acid sequences","volume":"78","author":"Hopp","year":"1981","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","first-page":"181","article-title":"The antigenic index: A novel algorithm for predicting antigenic determinants","volume":"4","author":"Jameson","year":"1988","journal-title":"Comput. Appl. Biosci."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Cosic, I. (1997). The Resonant Recognition Model of Macromolecular Bioactivity: Theory and Applications, Birkh\u00e4user.","DOI":"10.1007\/978-3-0348-7475-5"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1111\/j.1432-1033.1987.tb13692.x","article-title":"Prediction of \u201chot spots\u201d in SV40 enhancer and relation with experimental data","volume":"170","author":"Cosic","year":"1987","journal-title":"Eur. J. Biochem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Zelterman, D. (2010). Applied Linear Models with SAS, Cambridge University Press.","DOI":"10.1017\/CBO9780511778643"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2424","DOI":"10.3390\/ijms9122424","article-title":"Discovery of proteomic code with mRNA assisted protein folding","volume":"9","author":"Biro","year":"2008","journal-title":"Int. J. Mol. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1016\/0022-5193(82)90083-2","article-title":"Amino acid pairing","volume":"94","year":"1982","journal-title":"J. Theor. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1042\/bj2340679","article-title":"Binding of peptides that are specified by complementary RNAs","volume":"234","author":"Blalock","year":"1986","journal-title":"Biochem. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/0303-2647(92)90048-4","article-title":"Periodical changes of amino acid reactivity within the genetic code","volume":"27","author":"Siemion","year":"1992","journal-title":"Biosystems"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"507","DOI":"10.2174\/1389203043379413","article-title":"The problem of amino acid complementarity and antisense peptides","volume":"5","author":"Siemion","year":"2004","journal-title":"Curr. Protein Peptide Sci."},{"key":"ref_23","first-page":"573","article-title":"On the genetic origin of complementary protein coding","volume":"71","year":"1998","journal-title":"Croat. Chem. Acta."},{"key":"ref_24","first-page":"379","article-title":"\u03b1-Melanotropin peptide: Structure and ligand\u2013receptor recognition","volume":"79","author":"Houra","year":"2006","journal-title":"Croat. Chem. Acta"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3389","DOI":"10.1093\/nar\/25.17.3389","article-title":"Gapped BLAST and PSI-BLAST: A new generation of protein database search programs","volume":"25","author":"Altschul","year":"1997","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/0039-9140(85)80035-7","article-title":"Calculation of equilibrium constants from multiwavelength spectroscopic data-I mathematical considerations","volume":"32","author":"Gampp","year":"1985","journal-title":"Talanta."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/0039-9140(85)80077-1","article-title":"Calculation of equilibrium constants from multiwavelength spectroscopic data-II: SPECFIT: Two user-friendly programs in basic and standard FORTRAN 77","volume":"32","author":"Gampp","year":"1985","journal-title":"Talanta."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1016\/0039-9140(86)80233-8","article-title":"Calculation of equilibrium constants from multiwavelength spectroscopic data-IV Model-free least-squares refinement by use of evolving factor analysis","volume":"33","author":"Gampp","year":"1986","journal-title":"Talanta"},{"key":"ref_29","unstructured":"Maeder, M., and Neuhold, Y.-M. (2007). Practical Data Analysis, Elsevier B.V."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/anie.200903998","article-title":"Optical thermophoresis quantifies buffer dependence of aptamer binding","volume":"49","author":"Baaske","year":"2010","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1089\/adt.2011.0380","article-title":"Molecular interaction studies using microscale thermophoresis","volume":"9","author":"Wienken","year":"2011","journal-title":"Assay Drug Dev. Technol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Wienken, C.J., Baaske, P., Rothbauer, U., Braun, D., and Duhr, S. (2010). Protein-binding assays in biological liquids using microscale thermophoresis. Nat. Commun., 1.","DOI":"10.1038\/ncomms1093"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1073\/pnas.82.5.1372","article-title":"Similarity between the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA","volume":"82","author":"Bost","year":"1985","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1042\/bj2820773","article-title":"Design and recognition properties of a hydropathically complementary peptide to human interleukin 1\u03b2","volume":"282","author":"Fassina","year":"1992","journal-title":"Biochem. J."},{"key":"ref_35","first-page":"206","article-title":"Recombinant protein purification using complementary peptides as affinity tags","volume":"29","author":"Targovnik","year":"2012","journal-title":"Biotechnology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1002\/1439-7633(20020104)3:1<86::AID-CBIC86>3.0.CO;2-L","article-title":"Inhibition of \u03b2-amyloid aggregation and neurotoxicity by complementary (antisense) peptides","volume":"3","author":"Heal","year":"2002","journal-title":"ChemBioChem"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1002\/1439-7633(20020104)3:1<76::AID-CBIC76>3.0.CO;2-N","article-title":"Mechanistic investigation into complementary (antisense) peptide mini-receptor inhibitors of cytokine interleukin-1","volume":"3","author":"Heal","year":"2002","journal-title":"ChemBiochem"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1016\/0361-9230(86)90219-4","article-title":"Bovine pineal antireproductive tripeptide binds to luteinizing hormone-releasing hormone: A model for peptide modulation by sequence specific peptide interactions?","volume":"17","author":"Westall","year":"1986","journal-title":"Brain Res. Bull."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/(SICI)1098-2787(1997)16:1<1::AID-MAS1>3.0.CO;2-L","article-title":"Studying noncovalent protein complexes by electrospray ionization mass spectrometry","volume":"16","author":"Loo","year":"1997","journal-title":"Mass Spectrom. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1217","DOI":"10.1016\/j.molimm.2004.05.011","article-title":"De novo design of complementary (antisense) peptide mini-receptor inhibitor of interleukin 18 (IL-18)","volume":"41","author":"Bhakoo","year":"2004","journal-title":"Mol. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.ymeth.2012.12.005","article-title":"Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions","volume":"59","author":"Seidel","year":"2013","journal-title":"Methods"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Jerabek-Willemsen, M., Andr\u00e9, T., Wanner, R., Roth, H.M., Duhr, S., Baaske, P., and Breitsprecher, D. (2014). Microscale thermophoresis: Interaction analysis and beyond. J. Mol. Struct.","DOI":"10.1016\/j.molstruc.2014.03.009"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"10656","DOI":"10.1002\/anie.201204268","article-title":"Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding","volume":"51","author":"Seidel","year":"2012","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1038\/nature13116","article-title":"Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1","volume":"507","author":"Parker","year":"2014","journal-title":"Nature"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1006\/meth.1999.0927","article-title":"Optical spectroscopy in studies of antibody-hapten interactions","volume":"20","author":"Tetin","year":"2000","journal-title":"Methods"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1038\/2302","article-title":"NMR structure of human erythropoietin and a comparison with its receptor bound conformation","volume":"5","author":"Cheetham","year":"1998","journal-title":"Nat. Struct. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1166","DOI":"10.1093\/bioinformatics\/bts091","article-title":"Unipro UGENE: A unified bioinformatics toolkit","volume":"28","author":"Okonechnikov","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_48","unstructured":"STATISTICA Version 8.0. Available online:http:\/\/www.statsoft.com."},{"key":"ref_49","unstructured":"D\u00f6mling, A. (2013). Protein-Protein Interactions in Drug Discovery, Wiley-VCH."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"55","DOI":"10.2174\/138161208783330727","article-title":"Small molecule complementarity as a source of novel pharmaceutical agents and combination therapies","volume":"14","author":"Dillon","year":"2008","journal-title":"Curr. Pharm. Des."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/6\/9209\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:11:47Z","timestamp":1760217107000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/15\/6\/9209"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,5,26]]},"references-count":50,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2014,6]]}},"alternative-id":["ijms15069209"],"URL":"https:\/\/doi.org\/10.3390\/ijms15069209","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,5,26]]}}}