A combinatorial method to isolate short ribozymes from complex ribozyme libraries

Arriola, Joshua T.; Müller, Ulrich F.

doi:10.1093/nar/gkaa834

Cited by 5 publications

(5 citation statements)

References 49 publications

(57 reference statements)

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“…This library is usually generated by solid-phase synthesis, although mutagenic PCR can also be used when lower rates of mutagenesis (on the order of 1% per position) are desired [ 26 ]. The synthetic protocol can also be modified in various ways to incorporate deletions [ 27 , 28 ]. Selections using such libraries often yield variants with improved biochemical properties, and also provide valuable information about the sequence requirements and secondary structure of the motif [ 15 , 16 , 19 , 20 ].…”

Section: Discussionmentioning

confidence: 99%

Secondary Structure Libraries for Artificial Evolution Experiments

Sgallová

Curtis

2021

Molecules

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Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

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Section: Discussionmentioning

confidence: 99%

Secondary Structure Libraries for Artificial Evolution Experiments

Sgallová

Curtis

2021

Molecules

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“…Another advantage of reselection experiments is that they facilitate identification of the minimized core of a motif (here defined as the smallest construct needed for function). This is an important step because functional sequences isolated from random sequence libraries often contain nonessential regions that can be deleted or replaced with short spacers or loops (Figure 1d) [3,30,32–33,36–37] . Although such regions can sometimes also be identified making random deletions, minimization is usually greatly facilitated by data from reselection experiments (Figure 1d).…”

Section: How To Optimize a Functional Nucleic Acidmentioning

confidence: 99%

“…This is an important step because functional sequences isolated from random sequence libraries often contain nonessential regions that can be deleted or replaced with short spacers or loops (Figure 1 d). [ 3 , 30 , 32 , 33 , 36 , 37 ] Although such regions can sometimes also be identified making random deletions, minimization is usually greatly facilitated by data from reselection experiments (Figure 1 d). Minimized variants of functional motifs are less expensive to synthesize, tend to be better behaved from a kinetic perspective, and are often more amenable to high‐resolution structural analysis.…”

Section: How To Optimize a Functional Nucleic Acidmentioning

confidence: 99%

Pushing the Limits of Nucleic Acid Function

Curtis

2022

Chemistry A European J

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For many decades it was thought that information storage and information transfer were the main functions of nucleic acids. However, artificial evolution experiments have shown that the functional potential of DNA and RNA is much greater. Here I provide an overview of this technique and highlight recent advances which have increased its potency. I also describe how artificial evolution has been used to identify nucleic acids with extreme functions. These include deoxyribozymes that generate unusual products such as light, tiny motifs made up of fewer than ten nucleotides, ribozymes that catalyze complex reactions such as RNA polymerization, information‐rich sequences that encode overlapping ribozymes, motifs that catalyze reactions at rates too fast to be followed by manual pipetting, and functional nucleic acids which are active in extreme conditions. Such motifs highlight the limits of our knowledge and provide clues about as of yet undiscovered functions of DNA and RNA.

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“…[138][139][140][141] In addition, T7 system has been used extensively to produce antisense RNA or RNA ribozyme that plays a critical role in gene the rapy. [17,86,[142][143][144]…”

Section: Gene Therapymentioning

confidence: 99%

Construction and Application of Orthogonal T7 Expression System in Eukaryote: An Overview

Wang

Yan

et al. 2022

Advanced Biology

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to its high efficiency and yield for protein synthesis. [12] Among the commonlyused protein expression systems in E. coli (Table 1), T7 expession system prepared more than 90% of proteins in protein data bank (PDB) due to its high yield and translation efficiency for target protein. [13] The T7 system had been applied extensively for target protein and mRNA synthesis in prokaryote, eukaryote, and cell free system. [2] UP to now, it was also becoming an enabling tool for on-going synthetic biology, metabolic engineering, biomedicine, enzyme engineering, and so on. [14][15][16][17] Although T7 expression system had been developed successfully in various prokaryotes, [2] the construction of T7 expression system in eukaryote was still on-going, which attributed to the innate feature of transcription-translation system in eukaryote, for example, in prokaryote, the transcription was coupled with translation, while in eukaryote, mRNA was synthesized and modified in nucleus, then exporting to cytoplasm for protein synthesis. Thus far, there was no systematic and complete review about T7 expression system construction in eukaryote. In the present paper, the development of T7 expression system in eukaryote was reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine preparation and gene therapy. We also discussed the innate challenges of T7 expression system construction in eukaryote and its recent development in vaccine production and gene therapy. Animal Mammalian CellsDue to the pivotal role in life science research, clinical research, gene therapy, and vaccine production, mammalian cells were first selected as the eukaryotic host to construct T7 expression system; so far, mammalian cells have experienced a deep and systematic study on the construction of T7 expression system. In 1986, a vaccinia/T7 hybrid virus expression system (VT7 expression system) was constructed in CV-1 monkey kidney cells, in which two expression vectors co-infected host cells, including a recombinant vaccinia virus containing vaccinia promoter-driven T7 RNAP and a plasmid containing T7 promoter-driven target gene (Figure 1A). [26] Vaccinia is a cytoplasmic DNA virus, that is, the genome DNA of vaccinia exists in host cytoplasm. Vaccinia genome encodes all replication andThe T7 system is an orthogonal transcription-system, which is characterized by simplicity, higher efficiency, and higher processivity, and it is used for protein or mRNA synthesis in various biological-systems. In comparison with prokaryotes, the construction of the T7 expression system is still ongoing in eukaryotes, but it shows greatly applicable prospects. In the present paper, development of T7 expression system construction in eukaryotes is reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine production and gene therapy. In addition, the...

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A combinatorial method to isolate short ribozymes from complex ribozyme libraries

Cited by 5 publications

References 49 publications

Secondary Structure Libraries for Artificial Evolution Experiments

Secondary Structure Libraries for Artificial Evolution Experiments

Pushing the Limits of Nucleic Acid Function

Construction and Application of Orthogonal T7 Expression System in Eukaryote: An Overview

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