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[Affinity modification of proteins, binding nucleic acids in mammalian cells, by alkylating derivatives of
Abstract:
Using the 32P-labelled alkylating oligonucleotide derivative (pT)16, the oligonucleotide interaction with mammalian cells has been studied. The majority of fibroblastoid cell lines tested in this study (COS-1, vero, L-671, Ag 17-1, CHO, B7) as well as mouse hepatocytes were found to contain proteins specifically interacting with oligonucleotides. Cells of Ag 17-1 and COS-1, apart from the 79 kDa protein specific for all fibroblast lines, contained also a protein with a molecular mass of 83 kDa. In BALB/c mouse hepatocytes the 83 kDa protein is the major oligonucleotide binding protein. Analysis of concentration dependencies of specific modification of receptor proteins in liver cells has made it possible to determine the values of constants for the oligonucleotide derivatives binding to the protein. The binding constant for the alkylating oligonucleotide derivative pT16 and the corresponding phosphothioate oligonucleotide has been found to be equal to 5 x 10(6) M-1.
Insights
Mammalian cells, including fibroblasts and hepatocytes, possess specific proteins that bind to oligonucleotide derivatives. Researchers identified key binding proteins and quantified their interaction constants, revealing insights into cellular uptake mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Context:
- Oligonucleotide therapeutics require understanding cellular interactions.
- Specific protein binding influences oligonucleotide uptake and efficacy.
- Mammalian cells exhibit diverse protein expression profiles.
Purpose:
- To investigate the interaction of alkylating oligonucleotide derivatives with mammalian cells.
- To identify and characterize specific oligonucleotide-binding proteins in various cell lines and primary hepatocytes.
- To quantify the binding affinity of oligonucleotide derivatives to cellular proteins.
Summary:
- Studies using a 32P-labelled alkylating oligonucleotide derivative, (pT)16, revealed specific protein interactions in multiple mammalian cell lines (COS-1, vero, L-671, Ag 17-1, CHO, B7) and mouse hepatocytes.
- A 79 kDa protein was identified as common to fibroblastoid cells, while an 83 kDa protein was prominent in Ag 17-1, COS-1 cells, and BALB/c mouse hepatocytes.
- Binding constants were determined for the alkylating oligonucleotide derivative and its phosphothioate counterpart, with the binding constant for pT16 found to be 5 x 10^6 M^-1.
Impact:
- Identifies specific cellular targets for oligonucleotide delivery.
- Provides quantitative data on oligonucleotide-protein binding affinities.
- Contributes to the rational design of oligonucleotide-based drugs.