5′-Derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1

• Published in: FEBS letters 1991-04, Vol.281 (1), p.111-113
• Main Authors: Lokhova, I.A., Nevinsky, G.A., Godovikova, T.S., Ivanova, E.M., Koshkin, A.A., Sergeev, D.S., Frolova, E.I., Rudenko, N.K., Khomov, V.V., Kavsan, V.M., Zarytova, V.F., Lavrik, O.I.
• Format: Article
• Language: English
• Subjects: 5′-Derivatives d(pT) 8; Affinity; AMV reverse transcriptase; AMV-RT; Analytical, structural and metabolic biochemistry; Avian Myeloblastosis Virus - enzymology; Biological and medical sciences; derivatives of ethidium, N-(2-hydroxyethyl)-phenazinium, hemin, cholesterol and daunomycin in d(pT)8 analogs, respectively; DNA Polymerase I - metabolism; DNA Replication; Enzymes and enzyme inhibitors; Et-Phn-,Hem-, Chs-, Dnm; Fundamental and applied biological sciences. Psychology; Kinetics; Klenow fragment; Klenow fragment of E. coli DNA polymerase 1; Oligonucleotide Probes - chemical synthesis; Oligonucleotide Probes - metabolism; Peptide Fragments - metabolism; reverse transcriptase from avian myeloblastosis virus; RNA-Directed DNA Polymerase - metabolism; Structure-Activity Relationship; Transferases
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)80371-9

The K m and V max values for d(pT) 8 and its derivatives containing various 5′-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT) 8 of intercalators such as phenazinium, ethidium and daunomycin residues in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the K m values were 3 and 5 times higher than those for d(pT) 8. Compared to d(pT) 8, the affinity of FK to all the above analogs was 2.3—3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5′-end residues on the V max values of d(pT) 8 was small and ranged from 44% to 120% of that for d(pT) 8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5′-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.