Сборник содержит материалы научной мультиконференции «Биотех- нология медицине будущего»

Microbial production of recombinant RNAs for therapy and environmental control
Stepanov V.G.
1,2
, Liu Y.
2,3
, Willson R.C.
2
, Karpova G.G.
1
, Fox G.E.
2
1
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia 
2
University of Houston, Houston, USA 
3
Jiangsu Academy of Agricultural Sciences, Nanjing, China
Recent advances in RNA research create a demand for cost-effective production of RNA 
aptamers, antisense oligoribonucleotides, ribozymes, and other RNAs of practical importance. 
As of now, the majority of RNAs are made by either automated multistep chemical synthesis 
or enzymatic template-directed polymerization. Unfortunately, a sustained large scale RNA 
production by any of these methods remains too expensive to justify RNA use for anything 
else than selected medical applications where it can generate sufficient revenue to cover high 
manufacturing costs.
Hereby we present an integrated approach to cost-effective RNA production based on 
expression of recombinant RNAs in an appropriate bacterial host. Our method combines 
(1) a bacterial strain with genome-encoded phage RNA polymerase, (2) a plasmid vector 
which harbors an artificial RNA expression unit transcribed by the phage RNA polymerase to
a processable RNA scaffold fused with the RNA of interest, (3) a cell cultivation procedure which 
relies on selective stimulation of the phage RNA polymerase-transcribed gene expression to 
achieve high yields of the RNA chimera, and (4) a high-throughput RNA isolation and purifi-
cation protocol with minimum reliance on expensive equipment and toxic chemicals. Two 
tested RNA scaffolds were derived from small stable RNAs, 
Vibrio proteolyticus
5S rRNA and 
peptidyl transferase domain of 
Thermus thermophilus
23S rRNA. Each scaffold plays a dual 
role as a recognition element for the host enzymes which convert the primary transcript into 
mature RNA, and as a protective module that reduces polyadenylation and exoribonuclease 
trimming of the processed RNA and may provide some degree of protection against endoribo-
nucleases by hindering their access to the inserted cargo RNA. Increased intracellular accumu-
lation of the recombinant RNA chimera is achieved by selective suppression of the host RNA, 
DNA and protein synthesis using appropriate antibiotics added in mid-log phase of culture 
growth. After cell harvesting, RNA is isolated using neutral non-toxic lysis solution containing 
urea, SDS and EDTA as major acting agents. Purification of the recombinant RNA proceeds 
through fractional precipitation with CaCl2 and ethanol, and selective RNA capture by aggre-
gated denatured lysozyme. Finally, the RNA chimera is either cleaved to release the inserted 
RNA cargo or left intact if it is compatible with functionality of the cargo.
The approach was successfully applied to the production of shRNAs, and is considered 
for the production of anti-viral and anti-bacterial aptamers and ribozymes, anti-thrombotic 
aptamers, and shRNA-based nematocides.

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Всероссийская мультиконференция с международным участием «Биотехнология – медицине будущего»
29 июня - 2 июля 2019 г., г. Новосибирск, Россия
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