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.

Aptamers against viral proteins as a tool to answer biological questions

2019 ◽  
Author(s):  
◽  
Carolina Camargo
Keyword(s):  
Lentiviral Vector ◽  
Rna Aptamers ◽  
Target Cells ◽  
University Of Missouri ◽  
Practical Applications ◽  
Intracellular Expression ◽  
The University ◽  
Further Development ◽  
Viral Surface

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] There are two experimental chapters in this dissertation in which the fundamental questions center around aptamers and viruses and how these two concepts interlace. The first experimental chapter (chapter two) seeks to utilize previously characterized aptamers against HIV-1 Reverse Transcriptase (RT) that will be delivered by a lentiviral vector and which intracellular expression from different human promoters was evaluated. The goal of this study was to identify important elements of vector design that will impact transgene expression in target cells. This study was based on the hypothesis that intracellular expression of RNA aptamers delivered by a lentiviral vector could offer a platform to enable adequate aptamer expression that would translate into viral suppression. And the second experimental chapter (chapter four) describes an in vitro 2'FY-RNA selection against Filoviral glycoproteins and outlines three different strategies that were followed to achieve selection of specific aptamers. Aptamers described in this chapter were able to recognize Ebolavirus glycoprotein ectodomain as well as in its native conformation displayed on the viral surface. Taking the observations obtained in this dissertation, aptamer technology could be expanded into further development for practical applications.

Advances in aptamer evolution and engineering

2016 ◽  
Author(s):  
◽  
Khalid Kamal Alam
Keyword(s):  
High Throughput Sequencing ◽  
Target Selection ◽  
Three Dimensional ◽  
Rna Aptamers ◽  
Hiv Reverse Transcriptase ◽  
University Of Missouri ◽  
Selection Approach ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.

scholarly journals Bioinspired genotype–phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins

2015 ◽  
Vol 5 (4) ◽  
pp. 20150035 ◽  
Author(s):  
Liisa D. van Vliet ◽  
Pierre-Yves Colin ◽  
Florian Hollfelder
Keyword(s):  
Synthetic Biology ◽  
Directed Evolution ◽  
Enzyme Catalysis ◽  
Darwinian Evolution ◽  
Oil Droplets ◽  
Shell Beads ◽  
Cellular Compartmentalization ◽  
In Cells

The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments—water-in-oil droplets and gel-shell beads—for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

scholarly journals Synthetic Life with Alternative Nucleic Acids as Genetic Materials

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3483
Author(s):  
Peng Nie ◽  
Yanfen Bai ◽  
Hui Mei
Keyword(s):  
Nucleic Acids ◽  
Genetic Information ◽  
Structural Parameters ◽  
Living Cells ◽  
Information Storage ◽  
Chemically Modified ◽  
Synthetic Life ◽  
Living Organisms

DNA, the fundamental genetic polymer of all living organisms on Earth, can be chemically modified to embrace novel functions that do not exist in nature. The key chemical and structural parameters for genetic information storage, heredity, and evolution have been elucidated, and many xenobiotic nucleic acids (XNAs) with non-canonical structures are developed as alternative genetic materials in vitro. However, it is still particularly challenging to replace DNAs with XNAs in living cells. This review outlines some recent studies in which the storage and propagation of genetic information are achieved in vivo by expanding genetic systems with XNAs.

scholarly journals Amplification of RNA by an RNA polymerase ribozyme

2016 ◽  
Vol 113 (35) ◽  
pp. 9786-9791 ◽  
Author(s):  
David P. Horning ◽  
Gerald F. Joyce
Keyword(s):  
Nucleic Acids ◽  
Rna Polymerase ◽  
Genetic Information ◽  
Rna World ◽  
In Vitro Evolution ◽  
Rna Molecules ◽  
Encoded Proteins ◽  
Rna Genes ◽  
Functional Rna

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins.

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.

scholarly journals The Old and New RNA World

2014 ◽  
Vol 83 (4) ◽  
pp. 441-448
Author(s):  
Zofia Szweykowska-Kulińska
Keyword(s):  
Gene Expression ◽  
Genetic Information ◽  
Environmental Changes ◽  
Rna World ◽  
Chromatin Condensation ◽  
Feedback Regulation ◽  
Information Storage ◽  
Rna Molecules ◽  
Metabolic Reactions ◽  
Almost All

Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment) approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we live in a New RNA World.

scholarly journals Cofactors are Remnants of Life’s Origin and Early Evolution

2021 ◽  
Vol 89 (3) ◽  
pp. 127-133 ◽  
Author(s):  
Aaron D. Goldman ◽  
Betul Kacar
Keyword(s):  
Genetic Information ◽  
Evolutionary Biology ◽  
Active Sites ◽  
Rna World ◽  
Genetic System ◽  
Early Evolution ◽  
Modern Biology ◽  
Iron Sulfur Cluster ◽  
Adenosyl Methionine ◽  
Precursor Rna

AbstractThe RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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