Substrate for detection of mammalian 5-C-DNA methyltransferase

DETAILED DESCRIPTION OF THE INVENTION "Oligomeric DNA" or ODN, as used herein, means a DNA having from 10 to 75 "purine or pyrimidine bases" connected in a series of nucleotides which are deoxyribonucleotides or analogs of deoxyribonucleotides with chemically modified sugars or linkages between sugars.
Purine or pyrimidine bases as used herein mean adenine (A), cytosine (C), guanine (G), thymine (T), and analogs of C (5-methyl-cytosine, 5-fluoro-cytosine, 5-bromo-cytosine, etc.
) that have been chemically modified so as to continue to be chemically acceptable into a synthetic chain of deoxyribonucleic acid or deoxyribonucleic acid analogs.
"5-C-DNA methyl transferase" means DNA 5-cytosine methyl transferase for methylation of cytosine to form a 5-methyl-2'-deoxycytosine residue in DNA.
To obtain DNA analogs which are highly specific for mammalian DNA methyltransferase, we have artificially constructed ODN analogs of DNA which contain CG sequences that have the intrinsic potential to be recognized and methylated by DNA methyltransferases.
In general, we have chosen ODN constructs which are 24 bases in length, or 24mers, but our invention can also apply to ODNs that are shorter and/or longer than 24 bases in length e.
g.
, 10 to 75 and preferably 12 to 50 bases in length.
We have examined the activity of these ODNs as substrates for DNA methyltransferase, and have identified single-stranded as well as double-stranded constructs of DNA which are excellent substrates of DNA methyltransferase.
Four examples of DNA constructs are shown in Table 1.
They are labeled Sense (S), Antisense (AS), Methylated Antisense 1 (M1) and Methylated Antisense 2 (M2).
The mammalian DNA methyltransferase substrate activity of these 4 single-stranded ODNs is shown in Table 2.
It can be seen that M2 has exceptional substrate activity, especially in comparison with S and AS, which were designed to have an optimal number of potentially methylatable CG sites available.
In contrast to the remarkable substrate activity of M