scholarly journals Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

2021 ◽  
Author(s):  
Alicia Climent Catala ◽  
Thomas E Ouldridge ◽  
Guy-Bart V Stan ◽  
Wooli Bae
Keyword(s):  
Nucleic Acid ◽  
Synthetic Biology ◽  
Dna Sequences ◽  
Transcriptional Activity ◽  
Green Light ◽  
Rna Aptamers ◽  
Toggle Switch ◽  
Mutual Inhibition ◽  
T7 Promoter

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers ca- pable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 in- hibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like con- ditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control ele- ment for nucleic acid networks in synthetic biology applications.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

Ribozymes and aptamers in the RNA world, and in synthetic biology

2016 ◽  
Author(s):  
◽  
Raghav Raj Poudyal
Keyword(s):  
Synthetic Biology ◽  
Genetic Information ◽  
Rna World ◽  
Information Storage ◽  
Rna Aptamers ◽  
Darwinian Evolution ◽  
University Of Missouri ◽  
Biologically Relevant ◽  
Functional Rnas

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The RNA world hypothesis postulates that Ribonucleic Acids (RNA) may have provided functions of catalysis and genetic information storage during the origin of life on earth. An RNA based life is hypothesized to have undergone Darwinian evolution to ultimately lead into extant biology, where DNA is used as the repository for genetic information and proteins are used as biological catalysts. The discovery of functional RNAs such as catalytic RNAs, regulatory RNAs, and ligand-binding RNA aptamers further strengthen this hypothesis. These functional RNAs are also used as tools for synthetic biology and therapeutics. This work highlights strategies used by RNA enzymes (Ribozymes) for catalysis of chemical reactions, and explores new chemistries catalyzed by ribozymes. We also engineered an in vitro evolved ribozyme to control activities of other functional RNA molecules. Finally, this work explores innovative approaches to discover new RNA enzymes that catalyze biologically relevant reactions. Findings from these studies have revealed potential roles of RNA enzymes during the primordial earth, and also opened doors to build RNA-based tools that regulate biological processes.

scholarly journals Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005 ◽  
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

scholarly journals Synthetic bionanotechnology: synthetic biology finds a toehold in nanotechnology

2019 ◽  
Vol 3 (5) ◽  
pp. 507-516 ◽  
Author(s):  
Alexander A. Green
Keyword(s):  
Nucleic Acid ◽  
Synthetic Biology ◽  
Self Assembly ◽  
Cell Function ◽  
Genetically Engineered ◽  
Molecular Networks ◽  
Network Synthesis ◽  
Network Function

Enabled by its central role in the molecular networks that govern cell function, RNA has been widely used for constructing components used in biological circuits for synthetic biology. Nucleic acid nanotechnology, which exploits predictable nucleic acid interactions to implement programmable molecular systems, has seen remarkable advances in in vitro nanoscale self-assembly and molecular computation, enabling the production of complex nanostructures and DNA-based neural networks. Living cells genetically engineered to execute nucleic acid nanotechnology programs thus have outstanding potential to significantly extend the current limits of synthetic biology. This perspective discusses the recent developments and future challenges in the field of synthetic bionanotechnology. Thus far, researchers in this emerging area have implemented dozens of programmable RNA nanodevices that provide precise control over gene expression at the transcriptional and translational levels and through CRISPR/Cas effectors. Moreover, they have employed synthetic self-assembling RNA networks in engineered bacteria to carry out computations featuring up to a dozen inputs and to substantially enhance the rate of chemical synthesis. Continued advancement of the field will benefit from improved in vivo strategies for streamlining nucleic acid network synthesis and new approaches for enhancing network function. As the field matures and the complexity gap between in vitro and in vivo systems narrows, synthetic bionanotechnology promises to have diverse potential applications ranging from intracellular circuits that detect and treat disease to synthetic enzymatic pathways that efficiently produce novel drug molecules.

scholarly journals RNA strand invasion activity of the Polycomb complex PRC2

2019 ◽  
Author(s):  
Célia Alecki ◽  
Victoria Chiwara ◽  
Lionel A. Sanz ◽  
Daniel Grau ◽  
Osvaldo Arias Pérez ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Epigenetic Regulation ◽  
Dna Sequences ◽  
Polycomb Group ◽  
Epigenetic Mechanism ◽  
Biological Information ◽  
Loop Formation ◽  
Strand Invasion ◽  
Nucleic Acid Structures

AbstractEpigenetic regulation is conveyed through information encoded by specific chromatin features. Non-canonical nucleic acid structures could in principle also convey biological information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) proteins form memory of transient transcriptional repression events that is necessary for development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, active, or unengaged states 1,2. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, PcG protein complexes modify chromatin to maintain gene repression 1,3,4. However, PcG proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA hybridizes to its complementary DNA and displaces the other DNA strand 5, form at many PREs in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-loops. Our results suggest a new mechanism for targeting PcG function through R-loop formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

Use of reflected green light for specific identification of islets in vitro after collagenase isolation

Diabetes ◽  
1979 ◽  
Vol 28 (6) ◽  
pp. 612-613 ◽  
Author(s):  
E. H. Finke ◽  
P. E. Lacy ◽  
J. Ono
Keyword(s):  
Green Light ◽  
Specific Identification

In vitro evaluation of hexitol nucleic acid antisense oligonucleotides

1999 ◽  
Author(s):  
Arthur Van Aerschot ◽  
Mark Vandermeeren ◽  
Johan Geysen ◽  
Walter Luyten ◽  
Marc Miller ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
In Vitro Evaluation

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

Genetically Encoded RNA Nanodevices for Cellular Imaging and Regulation

Nanoscale ◽  
2021 ◽  
Author(s):  
Qikun Yu ◽  
Kewei Ren ◽  
Mingxu You
Keyword(s):  
Nucleic Acid ◽  
Cellular Imaging ◽  
Synthetic Dna

Nucleic acid-based nanodevices have been widely used in the fields of biosensing and nanomedicine. Traditionally, the majority of these nanodevices were first constructed in vitro using synthetic DNA or RNA...

Sign in / Sign up

Export Citation Format

Share Document