Nucleic Acids Research, Vol 24, Issue 11 2044-2052, Copyright © 1996 by Oxford University Press
S Moran, RX Ren, CJ Sheils, S Rumney 4th and ET Kool
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.
ARTICLES
Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis
Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
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