Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis.
We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.
Moran
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S
Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
Ren
R X
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Sheils
C J
CJ
Rumney
S
S
Kool
E T
ET
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003855:Deoxyribose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007211:Indoles / Q000138:chemical synthesis / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000138:chemical synthesis / Q000494:pharmacology*; D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011721:Pyrenes / Q000138:chemical synthesis / Q000494:pharmacology*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry; D013698:Templates, Genetic; D014764:Viral Proteins
C099895:1-naphthyl deoxynucleoside; C099896:1-pyrenyl deoxynucleoside; C099894:4-methylindole deoxynucleoside; D007211:Indoles; D009281:Naphthalenes; D009705:Nucleosides; D011721:Pyrenes; D014764:Viral Proteins; D003855:Deoxyribose; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I