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SELEX is Structure-Function Studies of Cytokines

#1038


Exploiting of SELEX Procedure in Structure-Function Studies of Recombinant Human Cytokines and Their Natural and Synthetic Antagonists in Order to Create Artificial Immunomodulating Medical Preparations

Tech Area / Field

  • MED-DRG/Drug Discovery/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology

Status
8 Project completed

Registration date
10.07.1997

Completion date
23.05.2003

Senior Project Manager
Pobedimskaya D D

Leading Institute
State Scientific Center of Genetics and Selection of Industrial Microorganisms (GosNIIGenetica), Russia, Moscow

Supporting institutes

  • Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany\nInstitute of Highly Pure Biopreparations, Russia, St Petersburg

Collaborators

  • Columbia University / Department of Chemistry / Neurology Department, USA, New York

Project summary

The project is devoted to one of the urging problems of today immunology and medicine. It includes: the structural and functional investigation of recombinant human cytokines and cytokine-like proteins; designing on this basis of modified cytokines and model peptides, artificial cytokine antagonists and modulators of separate cytokines' functions having in difference to their natural analogues, a narrower range of biological activity, in order to create the basis for construction of new immune modulating preparations intended for treating of a wide range of human diseases and states.

The experimental realization of the project is based on a new up-to-date approach, i.e. the SELEX (Systematic Evolution of Ligands by Exponential enrichment) procedure for a structural and functional analysis of biologically active macromolecules and construction of novel pharmaceutical preparations using short oligonucleotides. The SELEX experiment consists in the following. Using the available protocols for chemical synthesis of oligonucleotides with randomized sequences and amplification of initially negligible small amounts of specific DNA fragments with the polymerase chain reaction (PCR), it is possible to select from the originally non-homogeneous mixture of oligonucleotides with random sequences those oligonucleotides (aptamers) whose spatial structure would provide for high-affinity specific interaction with inpidual regions of the macromolecule studied and probably modification of its biological activity.

The unique combination of aptamers' features (small value of molecular size; tight three-dimensional structure; high affinity of complex formation between aptamers and macromolecules studied, which could exceed this value for complexes between proteins and antibodies) allows to suspect that exploiting of SELEX procedure, in addition to traditional approaches, leads to (1) modulating of biological activity of cytokines interacting with aptamers; (2) obtaining of new data concerning of spatial structures of cytokines' active centers; (3) developing of new approaches for designing and synthesis of rather short peptides modeling of separate cytokine functions; (4) gene-engineering construction of genes followed by obtaining of recombinant cytokines with modified pleiotropicity of biological activity. The results of these investigations could be used as a basis for creation of new generation of pharmaceutical preparations. Artificial aptamers, their complexes with recombinant cytokines, synthetic peptides and non-toxic analogs of cytokines, probably, could be applied as inpidual therapeutic preparations.

The aim of the project is exploiting of the SELEX procedure, in addition to traditional approaches, for structure-function investigation of recombinant human cytokines (hIL-1 a, hlL-l b, hlL-l ra, hIL-4, hIL-8, hIFN-a 2, hIFN-g, TNFa) and cytokine-like proteins (Caf1) in order to obtain aptamers that interact specifically with recombinant cytokines and can be applied for artificial antagonists and modulators of cytokine functions in vivo as well as to design model peptides, modified cytokines and complexes of cytokines with specific aptamers whose would have modified range of biological activities in comparison with native analogs. These investigations could be used as an initial step for creation of novel immunomodulating medical preparations. The above aim of the project can be achieved taking into account the great collaborative experience of the applicant institutions in genetic and protein engineering, structural and functional studies of cytokines proposed for research.

The main project goals


- exploiting of SELEX procedure for construction of aptamers specifically interacting with proposed recombinant cytokines;
- investigations of spatial structures of aptamers and their complexes with cytokines due to applying of wide range of physico-chemical, biological and immunological methods, and computer modeling;
- analysis of biological activities of specific aptamers and their complexes with recombinant cytokines;
- obtaining and studying of cytokines with modified range of biological activities, and designing and synthesis of model peptides having separate biological activities of native cytokines.

The main expected results


- due to exploiting of SELEX procedure single-stranded oligonucleotides, aptamers, would be obtained which interact high-efficiently with separate parts of human cytokines and modulate their biological functions;
- on the basis of methods of gene- and protein-engineering the chemical synthesis of model peptides would be provided as well as construction of bacterial strains producing of modified cytokines with narrowed pleiotropicity of biological activities followed by accumulation of recombinant proteins and model peptides and investigation of their physico-chemical and biological properties;
- the following information would be obtained:
- concerning of localization of active centers in cytokines, cytokine-like proteins and in corresponding receptor polypeptides;
- structural features of aptamers and their complexes with recombinant cytokines;
- mechanisms of modification of cytokine functions due to complex formation between protein molecules and aptamers;
- concerning of possibility of modeling of separate cytokine functions by rather short synthetic peptides;
- recommendations for application of model peptides, modified cytokines, aptamers and their complexes with cytokines, as prototypes of irnmunomodulating medical preparations of new generation for treating immunopathological diseases and states would be worked out.

Technical approach and methodology


- Specific aptamers to cytokines will be obtained by SELEX procedure of the chemically synthesized oligonucleotides with a random sequence, selecting high-affinity aptamers by immobilized cytokine column chromatography. Amplification of bound DNA aptamers will be done by asymmetric polymerase chain reaction (PCR), and reverse transcription will be performed for RNA oligonucleotides followed by amplification of double-stranded DNAs due to PCR with subsequent preparation of RNA-aptamers by transcription in vitro with phage T7 RNA polymerase. The nucleotide sequence of specific aptamers will be determined after their molecular cloning according to Sanger's procedure. Then it is proposed to use computer estimations of the secondary structure of single-stranded oligonucleotides and to analyze their three-dimensional structure by NMR.
- It is proposed to design modified recombinant cytokines by synthesis of model peptides, chemical synthesis of genes and/or site-specific mutagenesis of genes of natural cytokines with following cloning of these cistrones and their expression in bacterial cells, isolation and purification of recombinant polypeptides by standard methods of gene engineering, protein chemistry and chromatography.
- Investigation of biological characteristics of aptamers, their complexes with cytokines, model peptides and cytokines with a modified primary structure will be performed using panels of monoclonal antibodies, different physical and chemical methods, computer modeling as well as in specific tests to determine activity in vitro and exploiting of experimental models for appreciation of the influence on the immunity of laboratory animals.

Potential role of foreign collaborators

It is planned to provide joint investigations with foreign collaborators, scientific exchange of necessary preparations and information, joint publications, scientific visits to collaborators' laboratories, seminars and consultations.

Up to date the following potential scientific collaborators have agreed to participate in joint research project to contribute in solving its separate tasks:


- Prof, Dr. D. Fradelizi, Director, Investigator, Institute U-283, National Institute of Health and Medical Studies (INSERM), Paris, France. Tel.: (33) 43-26-11-52, Fax: (33) 43-26-11-56. Joint investigations in obtaining of monoclonal antibodies to cytokines, epitope mapping of cytokines and their complexes with aptamers; consultations, seminars, expertise.
- Dr. T. Korpela, Finnish-Russian Joint Biotechnology Laboratory, Department of Biochemistry, University of Turku, BioCity, 6th floor, SF-20520 Turku, Finland. Tel.: 358-2-333-80-62, Fax: 358-2-333-80-80.

Joint computer modeling of complexes of aptamers with cytokines; consultations, seminars, expertise.


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