458
Cell-free Translation Systems
sTC
yTC
DNA
with unnatural base
Unnatural
amino acid
CUs
mRNA
with unnatural
base
Transcription
Cell-free
translation
Protein
with unnatural
amino acid
(a)
(b)
N
N
N
N
R
N
H
H
O
H
N
O
H
N
N
H
R
isoG
isoC
N
N
N
N
R
H
N
N
H
O
R
y
S
S
H
H
Fig. 3
Structures of unnatural bases utilized in (a) cell-free translation for expansion of
genetic code and (b) design of transcription/translation coupled system using unnatural
nucleotides for incorporation of unnatural amino acids.
the incorporation of 3-chlorotyrocine was
successfully inserted into the desired po-
sition of protein utilizing base pairing
between 2-amino-6-(2-thienyl) purine (
s
)
and pyridin-2-1 (
y
) (Fig. 3b) as follows.
First, a DNA fragment in which the
s
-
nucleotide was located at the ±rst letter of
the desired codon was synthesized for the
synthesis of mRNA by transcription reac-
tion in the presence of the
y
-nucleotide.
Simultaneously, yeast tRNA
Tyr
with the
s
-nucleotide at the third position of the
anticodon was prepared by ligation of
RNA fragments and was aminoacylated
with 3-chlorotyrosine using tyrosyl-tRNA
synthetase. Using the
E. coli
cell-free trans-
lation system, 3-chlorotyrosine was suc-
cessfully incorporated at the desired po-
sition of a target protein. Although this
approach looks very expansible, techni-
cal dif±culties in site-speci±c introduction
of appropriate arti±cial nucleobases into
mRNA and tRNA remain to be solved for
the development of a concise and widely
distributed system.
2.3
Linkage between Genotype and Phenotype
Cell-free translation provides the potential
methodology for linkage between geno-
type and phenotype.
In vitro
screening of
DNA corresponding to RNAs and proteins
with certain functions is of signi±cant im-
portance in the ±eld of molecular biology.
In the case of functional RNA, such as ri-
bozymes, it is easy to convert to DNA using
the reverse transcriptase after the selection
procedure designed for the desired func-
tion. In contrast, we have no direct system
to convert from polypeptide to mRNA.
However, the complex formation of nu-
cleic acid and its protein product enables
one to perform pseudoreverse translation.
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