scholarly journals Secondary Structure Libraries for Artificial Evolution Experiments

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1671
Author(s):  
Ráchel Sgallová ◽  
Edward A. Curtis
Keyword(s):  
Secondary Structure ◽  
Sequence Space ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Random Mutagenesis ◽  
Nucleotide Composition ◽  
Artificial Evolution ◽  
Wide Range ◽  
Range Of Functions

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.

Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

2004 ◽  
Vol 76 (7-8) ◽  
pp. 1547-1561 ◽  
Author(s):  
R. Nutiu ◽  
Shirley Mei ◽  
Zhongjie Liu ◽  
Y. Li
Keyword(s):  
Dna Sequences ◽  
Rational Design ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Pair Potential ◽  
Dna Aptamers ◽  
Dna Molecules ◽  
Dna Enzymes ◽  
Fluorescence Signaling

Single-stranded DNA molecules with ligand-binding ability and catalytic function, referred to as DNA aptamers and DNA enzymes, respectively, are special DNA sequences isolated from random-sequence DNA libraries by “in vitro selection”. These two new classes of artificial DNA molecules have the potential of being used as molecular tools in a variety of innovative applications ranging from biosensing to gene regulation. Our laboratory is interested in engineering fluorescence-signaling DNA aptamers and DNA enzymes that can be widely exploited for detection-directed applications. In this article, we will first discuss our recent efforts on the rational design of a new class of signaling aptamers denoted “structure- switching signaling aptamers”, which report target binding by switching structures from DNA/DNA duplex to DNA/target complex. We will then describe the in vitro selection of fluorescence-signaling DNA enzymes that exhibit a synchronized catalysis-signaling capability by cleaving a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by closely spaced fluorophore-quencher pair. Potential utilities of these signaling DNA molecules will also be discussed.

scholarly journals In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

2020 ◽  
Vol 117 (11) ◽  
pp. 5741-5748 ◽  
Author(s):  
Travis Walton ◽  
Saurja DasGupta ◽  
Daniel Duzdevich ◽  
Seung Soo Oh ◽  
Jack W. Szostak
Keyword(s):  
Origin Of Life ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Bond Formation ◽  
Rna Sequences ◽  
Phosphodiester Bond ◽  
Complex Protein ◽  
The Origin Of Life ◽  
Group 2

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide–activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

2004 ◽  
Vol 44 (1) ◽  
pp. 27 ◽  
Author(s):  
M. Bajji ◽  
P. Bertin ◽  
S. Lutts ◽  
J-M. Kinet
Keyword(s):  
Durum Wheat ◽  
Somaclonal Variation ◽  
Drought Resistance ◽  
In Vitro Selection ◽  
Culture Media ◽  
Field Studies ◽  
Resistance Mechanisms ◽  
Wide Range ◽  
Triticum Durum Desf

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4�generations. Differences in electrolyte leakage, chlorophyll fluorescence (Fv/Fm), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4�parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

Synthesis of Random DNA Libraries for In Vitro Selection and Analysis of Their Nucleotide Composition

2019 ◽  
Vol 45 (6) ◽  
pp. 656-661 ◽  
Author(s):  
A. S. Davydova ◽  
O. A. Krasheninina ◽  
A. E. Tupikin ◽  
M. R. Kabilov ◽  
A. G. Venyaminova ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Nucleotide Composition ◽  
Dna Libraries

Structure-function investigation of a deoxyribozyme with dual chelatase and peroxidase activities

2004 ◽  
Vol 76 (7-8) ◽  
pp. 1537-1545 ◽  
Author(s):  
H.-W. Lee ◽  
D. J.-F. Chinnapen ◽  
D. Sen
Keyword(s):  
In Vitro Selection ◽  
Structural Model ◽  
Random Sequence ◽  
Structure And Function ◽  
Dna Library ◽  
Dna Molecule ◽  
Folded Structure ◽  
And Function ◽  
Porphyrin Metallation

PS2.M, an 18-nucleotide DNA molecule, has been shown to be a dual enzyme for porphyrin metallation and, when complexed with hemin, for peroxidation. To date, detailed information has not been available on either the actively folded structure of PS2.M or on the contribution of specific nucleotides within it toward the peroxidase activity. Here, we report a variety of experiments that probe the structure and function of PS2.M as well as of a number of point mutants of PS2.M. Based on these experiments, a structural model for the folding of PS2.M and the location of a functionally relevant hemin-binding site are proposed. A key finding is that PS2.M, originally obtained by in vitro selection from a random-sequence DNA library, is uniquely suited for its catalysis of peroxidation; all point mutants examined showed significantly poorer catalytic activity than PS2.M itself.

scholarly journals Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 18
Author(s):  
Janne Leivo ◽  
Markus Vehniäinen ◽  
Urpo Lamminmäki
Keyword(s):  
In Vitro Selection ◽  
Specific Binding ◽  
Binding Properties ◽  
Phage Displays ◽  
Synthetic Antibody ◽  
Wide Range ◽  
Antibody Libraries ◽  
Rapid Generation ◽  
Selection Of

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

scholarly journals Secondary Structure as a Functional Feature in the Downstream Region of Mammalian Polyadenylation Signals

2004 ◽  
Vol 24 (7) ◽  
pp. 2789-2796 ◽  
Author(s):  
Chunxiao Wu ◽  
James C. Alwine
Keyword(s):  
Secondary Structure ◽  
Secondary Structures ◽  
Loop Size ◽  
Alternative Structures ◽  
Cleavage Efficiency ◽  
Downstream Region ◽  
Wide Range ◽  
Functional Features ◽  
Polyadenylation Signals

ABSTRACT Secondary structure within the downstream region of mammalian polyadenylation signals has been proposed to perform important functions. The simian virus 40 late polyadenylation signal (SVLPA) forms alternate secondary structures in equilibrium. Their formation correlates with cleavage-polyadenylation efficiency (H. Hans and J. C. Alwine, Mol. Cell. Biol. 20:2926-2932, 2000; M. I. Zarudnaya, I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun, Nucleic Acids Res. 3:1375-1386, 2003), and oligonucleotides that disrupt the secondary structure inhibit in vitro cleavage. To define the important features of downstream secondary structure, we first minimized the SVLPA by deletion, forming a downstream region with fewer, and more stable, stem-loop structures. Specific mutagenesis showed that both stem stability and loop size are important functional features of the downstream region. Stabilization of the stem, thus minimizing alternative structures, decreased cleavage efficiency both in vitro and in vivo. This was most deleterious when the stem was stabilized at the base of the loop, constraining loop size by inhibiting breathing of the stem. The significance of loop size was supported by mutants that showed increased cleavage efficiency with increased loop size and vice versa. A loop of at least 12 nucleotides promoted cleavage; U richness in the loop also promoted cleavage and was particularly important when the stem was stabilized. A mutation designed to eliminate downstream secondary structure still formed many relatively weak alternative structures in equilibrium and retained function. The data suggest that although the downstream region is very important, its structure is quite malleable and is able to tolerate significant mutation within a wide range of primary and secondary structural features. We propose that this malleability is due to the enhanced ability of GU- and U-rich downstream elements to easily form secondary structures with surrounding sequences.

scholarly journals Identification of 56 Proteins Involved in Embryo–Maternal Interactions in the Bovine Oviduct

2020 ◽  
Vol 21 (2) ◽  
pp. 466 ◽  
Author(s):  
Charles Banliat ◽  
Guillaume Tsikis ◽  
Valérie Labas ◽  
Ana-Paula Teixeira-Gomes ◽  
Emmanuelle Com ◽  
...  
Keyword(s):  
Protein Interactions ◽  
High Resolution Mass Spectrometry ◽  
Bovine Embryo ◽  
Interacting Proteins ◽  
Cellular Processes ◽  
Wide Range ◽  
Range Of Functions ◽  
First Time

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4–5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4–6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

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