Incorporation of a complete set of deoxyadenosine and thymidine analogs suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase

Abstract

A complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.

Specific Inhibition of Influenza Virus RNA Polymerase and Nucleoprotein Gene Expression by Liposomally Encapsulated Antisense Phosphorothioate Oligonucleotides in MDCK Cells

“…The oligonucleotide derivatives were purified by reverse-phase HPLC chromatography. The extinction coefficients of the oligonucleotides were determined by calculating the theoretical extinction coefficients as the sum of the nucleosides, and multiplying with the experimentally determined enzymatic hypochromicity (Newman et al, 1990).…”

DNA Distortion and Base Flipping by the EcoRV DNA Methyltransferase

“…The EcoRV methylase adds CH 3 groups to the first dA in GATATC 1 targets (1) and forms a stable complex with this sequence in the presence of its co-factor, S-adenosyl-L-methionine (AdoMet), 2 or co-factor analogues such as sinefungin (2)(3)(4)(5). Tightest binding is observed with hemimethylated GATATC sequences (K d ϭ 13 nM), and both unmethylated (K d ϭ 46 nM) and dimethylated (K d ϭ 143 nM) sequences bind less strongly.…”

Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease

“…One of the commonest methods for evaluating proteinsubstrate interactions is to make alterations to the partners involved and observe the consequences. This has been widely carried out for protein amino acids using site-directed mutagenesis and for DNA with modified bases (11)(12)(13)(14)(15)(16). For DNA phosphates the most useful analogues are the phosphorothioates (17)(18)(19)(20)(21), illustrated in Fig.…”