Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 157

4.3.2 Enzymatic Synthesis of Oligoribonucleotides

4.3.2.1 Transcription by T7 RNA Polymerase. A powerful method of enzymatic RNA synthesis

makes use of the RNA polymerase (see Sections 3.6.2 and 10.7.2) from bacteriophage T7 to copy a syn-
thetic DNA template.^11 The template is prepared from two chemically synthesised oligodeoxyribonu-
cleotides. Upon annealing, a duplex is formed corresponding to the base-pairs17 to1 of the T7
promoter sequence. Position1 is the site of initiation of transcription, which in natural DNA would be
in a fully base-paired duplex. For short RNA transcripts of 10–60 residues, it is possible to use a bottom
strand that carries a single-stranded 5
-extension corresponding to the complement of the desired oligori-
bonucleotide. Transcription of this template in vitrowith T7 RNA polymerase and nucleoside triphos-
phates gives up to 40mol of transcript per micromole of template (Figure 4.15).
Unfortunately there are limitations to this method. There are significant variations in the yield of RNA
run-off transcripts, especially depending on the sequence from1 to5 in the template. In some cases there
can be a high proportion of abortively-initiated transcripts. Transcription of higher efficiency and reliabil-
ity is often obtained by the use of a fully double-stranded DNA template, either by chemical synthesis
of both strands or by transcription of a plasmid DNA where the desired sequence is cloned 3
- to a T7-
promoter and linearised by cutting with a restriction enzyme (see Section 5.3.1). Run-off transcription
takes place up to the end of the DNA duplex at the restriction site.
A second problem is that in some cases a non-template-encoded nucleotide may be added to the
oligoribonucleotide or the main product may be one nucleotide shorter than expected. An ingenious solu-
tion to this problem is to engineer the desired sequence within the plasmid 3
- to the T7-promoter and
flanked by other sequences which, when transcribed, fold into self-cleavage domains,^12 as for example for
the hammerhead (5
-flank) and hepatitis delta virus (3
-flank) ribozymes (see Section 7.6.2). During tran-
scription the transcribed RNA folds and cleaves itself to give unique 5
- and 3
-ends.
To obtain oligoribonucleotides lacking the 5
-triphosphate, whichever transcription method is used, it is
possible to initiate transcription by including in the reaction a high proportion of rGpG or the nucleoside
rG, which is incorporated at the 5
-end of the transcript.

4.3.2.2 Joining of Oligoribonucleotides. An RNA ligase from the bacteriophage T4 (RNA ligase 1)

catalyses the joining of a 5
-phosphate group of a donormolecule (minimum structure pNp) to a 3
-hydroxyl
group of an acceptoroligonucleotide (minimum structure NpNpN) (Figure 4.16).^13
The enzyme exhibits a high degree of preference for particular nucleotide sequences, favouring purines
in the acceptor and a pyrimidine at the 5
-terminus of the donor, although there are substantial variations
depending on the exact sequences of each. To prevent other possible ligation reactions, the acceptor

dTAATACGACTCACTATAG

ATTATGCTGAGTGATATCCCTCAGTACTAGCd

rpppGGGAGUCAUGAUCG

5'

3' 5'

3'

CH-17 -1+1

5' 3'

4NTPs

T7 promoter

T7 RNA polymerase

Figure 4.15 Use of T7 RNA polymerase to transcribe synthetic DNA templates