A Simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin

Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario (2014) A Simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin. International Journal of Molecular Sciences, 15 (6). pp. 9209-9223. ISSN 1422-0067

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Official URL: http://www.mdpi.com/1422-0067/15/6/9209

Abstract

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor 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'→5' direction and computational screening of potential paratope structures with BLAST ; Thirdly, we evaluated sense–antisense (epitope–paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, 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.

Item Type:

Article

Uncontrolled Keywords:

erythropoietin; antisense; peptide; binding; fluorescence; spectroscopy; thermophoresis; modeling

Subjects:

BIOMEDICINE AND HEALTHCARE > Pharmacy

Divisions:

NMR Center

Projects:

Project title	Project leader	Project code	Project type
Modelling of bioactive molecules and testing of their properties and activity (Modeliranje bioaktivnih molekula i ispitivanje njihovih svojstava i učinka)-	Nikola Štambuk	098-0982929-2524	MZOS

Depositing User:

Paško Konjevoda

Date Deposited:

02 Oct 2015 10:59

URI:

http://fulir.irb.hr/id/eprint/2142

DOI:

10.3390/ijms15069209