Recognition by restriction endonuclease EcoRI of deoxyoctanucleotides containing modified sugar moieties.
The deoxyribooctanucleotide d(G-G-A-A-T-T-C-C), containing the recognition sequence for EcoRI, d(G-A-A-T-T-C), and analogs containing modified sugar moieties were tested for their activity in cleavage with EcoRI. These analogs, with replacement in the third position from the 5' end, were synthesized using 9-beta-D-arabinosyladenine (aA), 2'-deoxy-2'-fluoroadenosine (Afl) and adenosine (rA). Duplex formation by the three analogs was confirmed by measurements of ultraviolet/temperature profiles. It was found that EcoRI cleaved these duplexes less efficiently than d(G-G-A-A-T-T-C-C). The adenosine-containing analog d(G-G)-rA-d(A-T-T-C-C) was cleaved much more slowly than d(G-G)-aA-d(A-T-T-C-C) and d(G-G-Afl-A-T-T-C-C). The corresponding ribooctamer G-G-A-A-U-U-C-C showed a higher melting temperature than the deoxyoctamers but its duplex was not cleaved by this enzyme. An analog with 2'-deoxy-2'-fluoroguanosine at the second position was cleaved by the endonuclease faster than the natural deoxyoctamer.
D001665:Binding Sites; D050260:Carbohydrate Metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI