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RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells
journal contribution
posted on 2023-12-11, 05:47 authored by Feiyang Zheng, Yoshinori Kawabe, Masamichi KamihiraRNA expression analyses can be used
to obtain various
information
from inside cells, such as physical conditions, the chemical environment,
and endogenous signals. For detecting RNA, the system regulating intracellular
gene expression has the potential for monitoring RNA expression levels
in real time within living cells. Synthetic biology provides powerful
tools for detecting and analyzing RNA inside cells. Here, we devised
an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered
gene expression activation by employing an inducible complex formation
strategy grounded in synthetic biology. This methodology connects
DNA-binding domains and transactivators through target RNA using RNA-binding
domains, including phage coat proteins. MS2 bacteriophage coat protein
fused with a transcriptional activator and PP7 bacteriophage coat
protein fused with the tetracycline repressor (tetR) can be bridged
by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional
activation. We generated recombinant CHO cells containing an inducible
GFP expression module governed by a minimal promoter with a tetR-responsive
element. Cells carrying the trigger RNA exhibited robust reporter
gene expression, whereas cells lacking it exhibited no expression.
GFP expression was upregulated over 200-fold compared with that in
cells without a target RNA expression vector. Moreover, this system
can detect the expression of mRNA tagged with aptamer tags and modulate
reporter gene expression based on the target mRNA level without affecting
the expression of the original mRNA-encoding gene. The RNA-triggered
gene expression systems developed in this study have potential as
a new platform for establishing gene circuits, evaluating endogenous
gene expression, and developing novel RNA detectors.
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obtain various informationmethodology connects dnaestablishing gene circuitswhereas cells lackinginducible transgene expressionencoding geneinside cellscells withoutcells carryingtranscriptional activatortranscriptional activationtetracycline repressorsynthetic biologyresponsive elementpp7 stemphysical conditionsoriginal mrnanew platformmrna taggedminimal promotergfp expressionfold comparedendogenous signalschemical environmentbinding domainsaptamer tags
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