Dataset Information

Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation.

ABSTRACT: Trans-regulating elements such as noncoding RNAs are crucial in modifying cells, and has shown broad application in synthetic biology, metabolic engineering and RNA therapies. Although effective, titration of the regulatory levels of such elements is less explored. Encouraged by the need of fine-tuning cellular functions, we studied key parameters of the antisense RNA design including oligonucleotide length, targeting region and relative dosage to achieve differentiated inhibition. We determined a 30-nucleotide configuration that renders efficient and robust inhibition. We found that by targeting the core RBS region proportionally, quantifiable inhibition levels can be rationally obtained. A mathematic model was established accordingly with refined energy terms and successfully validated by depicting the inhibition levels for genomic targets. Additionally, we applied this fine-tuning approach for 4-hydroxycoumarin biosynthesis by simultaneous and quantifiable knockdown of multiple targets, resulting in a 3.58-fold increase in titer of the engineered strain comparing to that of the non-regulated. We believe the developed tool is broadly compatible and provides an extra layer of control in modifying living systems.

SUBMITTER: Zhang R

PROVIDER: S-EPMC7921874 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation.

Zhang Ruihua R Zhang Yan Y Wang Jian J Yang Yaping Y Yan Yajun Y

Metabolic engineering communications 20210219

Trans-regulating elements such as noncoding RNAs are crucial in modifying cells, and has shown broad application in synthetic biology, metabolic engineering and RNA therapies. Although effective, titration of the regulatory levels of such elements is less explored. Encouraged by the need of fine-tuning cellular functions, we studied key parameters of the antisense RNA design including oligonucleotide length, targeting region and relative dosage to achieve differentiated inhibition. We determined ...[more]

PMID: 33717978

Similar Datasets

Project description:The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Dataset Information

Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation.

Publications

Development of antisense RNA-mediated quantifiable inhibition for metabolic regulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure