Crosslinking of double-stranded oligonucleotides containing O-methyl-substituted pyrophosphate groups to the HNF1 transcription factor in nuclear cell extract.
Probing of the HNF1 (hepatocyte nuclear factor I) DNA-binding region using a set of DNA duplexes containing pyrophosphate or O-methyl-substituted pyrophosphate internucleotide groups at different positions of the HNF1 recognition sequence was performed. The histidine-tagged HNF1/1-281 DNA binding domain and nuclear extract from rat liver were used. We showed that HNF1 from these species specifically binds to modified DNA duplexes. A correlation in binding affinity of both types of duplexes was detected. Crosslinking of the HNF1 DNA-binding domain and HNF1 in nuclear liver extract to DNA duplexes carrying O-methyl-substituted pyrophosphate groups was observed. The crosslinking efficiency of HNF1 in liver extract to substituted pyrophosphate-modified DNA duplex, containing a reactive internucleotide group between nucleotides G and T of the GT dinucleotide immediately 5' to the TAAT recognition sequence, amounts to 40% of the efficiency of non-covalent association. Nonspecific crosslinking of the reactive DNA duplexes to other components of nuclear extract was not observed. These results indicate that DNA duplexes carrying substituted pyrophosphate internucleotide groups can specifically bind and crosslink with DNA-binding proteins, especially transcription factors in crude preparations and could constitute a potential tool to control the expression of disease-causing genes.
Kuznetsova
S A
SA
Joint Laboratory GENSET-Laboratory of Nucleic Acid Chemistry, Moscow State University, Russia. kuznetsova@biorg.chem.msu.su
Clusel
C
C
Ugarte
E
E
Elias
I
I
Vasseur
M
M
Blumenfeld
M
M
Shabarova
Z A
ZA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006639:Histidine; D008099:Liver / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010710:Phosphates; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D006639:Histidine; D004247:DNA