Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

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.

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Secondary Structure Libraries for Artificial Evolution Experiments

Molecules ◽

10.3390/molecules26061671 ◽

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.

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Application of the Open qPCR Instrument for the in Vitro Selection of DNA Aptamers against Epidermal Growth Factor Receptor and Drosophila C Virus

ACS Combinatorial Science ◽

10.1021/acscombsci.7b00138 ◽

2018 ◽

Vol 20 (2) ◽

pp. 45-54 ◽

Cited By ~ 7

Author(s):

Tulsi Ram Damase ◽

Tanya A. Miura ◽

Christine E. Parent ◽

Peter B. Allen

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

In Vitro Selection ◽

Growth Factor Receptor ◽

Dna Aptamers ◽

Epidermal Growth ◽

Selection Of

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In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

Current Protocols in Chemical Biology ◽

10.1002/cpch.28 ◽

2017 ◽

Vol 9 (4) ◽

pp. 233-268 ◽

Cited By ~ 7

Author(s):

Annamaria Ruscito ◽

Erin M. McConnell ◽

Anna Koudrina ◽

Ranganathan Velu ◽

Christopher Mattice ◽

...

Keyword(s):

Small Molecule ◽

In Vitro Selection ◽

Dna Aptamers

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914367117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 5741-5748 ◽

Cited By ~ 2

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.

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In vitro selection of DNA aptamers against human osteosarcoma

Investigational New Drugs ◽

10.1007/s10637-021-01161-y ◽

2021 ◽

Author(s):

Khaliunsarnai Tsogtbaatar ◽

Diana A. Sousa ◽

Debora Ferreira ◽

Atakan Tevlek ◽

Halil Murat Aydın ◽

...

Keyword(s):

In Vitro Selection ◽

Dna Aptamers ◽

Human Osteosarcoma ◽

Selection Of

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Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2010.1491 ◽

2010 ◽

Vol 1 (1) ◽

pp. 3

Author(s):

Ivan B. Brukner ◽

Anne-Marie Larose ◽

Izabella Gorska-Flipot ◽

Maja Krajinovic ◽

Damian Labuda

Keyword(s):

Rational Design ◽

In Vitro Selection ◽

Clinical Performance ◽

Initial Step ◽

Detection Methods ◽

Ambient Temperatures ◽

Hybridization Probes ◽

Specificity And Sensitivity ◽

Hpv Types

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through i<em>n vitro </em>selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

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