Enzymatic processes for synthesizing RNA containing certain non-standard nucleotides
View Patent ↗This invention relates to processes that transcribe DNA molecules containing non-standard nucleotides using variants of T7 RNA polymerase to give RNA transcripts that contain their complementary non-standard nucleotides. Non-standard nucleotides pair during transcription using patterns of hydrogen bonding that are different from patterns that join the thymine-adenine and guanine-cytosine nucleobase pairs.
1. A process for synthesizing RNA containing one or more non-standard nucleotides, wherein said process
comprises contacting in aqueous solution
(a) a variant of T7 RNA polymerase that can accept non-standard nucleoside triphosphates that do not present electron density to the minor groove with
(b) a DNA template comprising a promoter for said variant, and
(c) nucleoside triphosphates that
comprise one or more independently selected heterocycles selected from the group consisting of
wherein M is an alkyl, alkenyl, or alkynyl substituent, either simple or functionalized, and R is the point of attachment of said heterocycle(s) to the ribose ring of said nucleoside triphosphate(s).
2. The process of claim 1 , wherein said variant to T7 RNA polymerase has amino acids replaced at individual sites in its polypeptide sequence, wherein said replacements comprise a replacement of tyrosine at position 639 by phenylalanine, a replacement of histidine at position 784 by alanine, and a replacement of proline at position 266 by leucine.
3. The process of claim 1 , wherein said nucleoside triphosphates comprise one or more independently selected heterocycles selected from the group consisting of
and M is an alkyl, alkenyl, or alkynyl substituent, either simple or functionalized, and R is the point of attachment of said heterocycle(s) to the ribose ring of said nucleoside triphosphate(s).
4. The process of claim 3 , wherein M is methyl.
5. The process of claim 1 , wherein said nucleoside triphosphates comprise one or more independently selected heterocycles selected from the group consisting of
and R is the point of attachment of said heterocycle(s) to the ribose ring of said nucleoside triphosphate(s).
6. The process of claim 1 , wherein said nucleoside triphosphate(s) comprise(s) the heterocycle
wherein R is the point of attachment of said heterocycle(s) to the ribose ring of said nucleoside triphosphate(s).