sb7b00115_si_001.pdf (496.84 kB)
Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells
journal contribution
posted on 2017-06-27, 15:19 authored by Sydney
E. Morris, Aaron W. Feldman, Floyd E. RomesbergTo bestow cells with
novel forms and functions, the goal of synthetic
biology, we have developed the unnatural nucleoside triphosphates
dNaMTP and dTPT3TP, which form an unnatural
base pair (UBP) and expand the genetic alphabet. While the UBP may
be retained in the DNA of a living cell, its retention is sequence-dependent.
We now report a steady-state kinetic characterization of the rate
with which the Klenow fragment of E. coli DNA polymerase
I synthesizes the UBP and its mispairs in a variety of sequence contexts.
Correct UBP synthesis is as efficient as for a natural base pair,
except in one sequence context, and in vitro performance
is correlated with in vivo performance. The data
elucidate the determinants of efficient UBP synthesis, show that the
dNaM-dTPT3 UBP is the first generally recognized
natural-like base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts
for the expansion of the genetic alphabet.