Stabilization of RNA hairpins using non-nucleotide linkers and circularization

Kiliszek, Agnieszka; Błaszczyk, Leszek; Kierzek, Ryszard; Rypniewski, W.

doi:10.1093/nar/gkx122

Cited by 8 publications

(7 citation statements)

References 45 publications

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“…A new band was observed to migrate faster than the linear form, which we referred as circular form (Figure 2 , lane 4). The faster migration of circular form compared to linear form is not uncommon, and likely caused by its more compact structure after cyclization ( 36 , 37 , 41 ). Importantly, we found no evidence of slower migration band above the linear form band that may indicate intermolecular click reaction caused by two linear L-Apt-4-1c (Figure 2 ), suggesting that our rational aptamer construct design and optimized click reaction conditions favor intramolecular cyclization.…”

Section: Resultsmentioning

confidence: 99%

“…The click reaction was performed based on the previously reported method with further optimization ( 36 ). The 5′-alkyne and 3′-azide labeled L-Apt.4-1c was synthesized by company.…”

Section: Methodsmentioning

confidence: 99%

“…Among the various chemical- or enzymatic-based cyclization methods, click chemistry reaction can provide a simple method to ligate nucleic acid molecules with high efficiency at mild reaction conditions ( 38 , 39 ). The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the best example for this kind of reactions ( 36 , 38 ). Azide and alkyne groups can generally be introduced to nucleic acids easily without affecting their biophysical properties.…”

Section: Introductionmentioning

confidence: 99%

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Circular L-RNA aptamer promotes target recognition and controls gene activity

Lyu

Zhao

et al. 2021

Nucleic Acids Research

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Rational design of aptamers to incorporate unnatural nucleotides and special chemical moieties can expand their functional complexity and diversity. Spiegelmer (L-RNA aptamer) is a unique class of aptamer that is composed of unnatural L-RNA nucleotides, and so far there are limited L-RNA aptamer candidates and applications being reported. Moreover, the target binding properties of current L-RNA aptamers require significant improvement. Here, using L-Apt.4-1c as an example, we develop a simple and robust strategy to generate the first circular L-RNA aptamer, cycL-Apt.4-1c, quantitatively, demonstrate substantial enhancement in binding affinity and selectivity toward its target, and notably report novel applications of circular L-RNA aptamer in controlling RNA–protein interaction, and gene activity including telomerase activity and gene expression. Our approach and findings will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications.

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Section: Resultsmentioning

confidence: 99%

“…The click reaction was performed based on the previously reported method with further optimization ( 36 ). The 5′-alkyne and 3′-azide labeled L-Apt.4-1c was synthesized by company.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Circular L-RNA aptamer promotes target recognition and controls gene activity

Lyu

Zhao

et al. 2021

Nucleic Acids Research

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“…To investigate whether the free ends of the RNA adopting a G-quadruplex structure are necessary for its binding to the hDicer PPC cassette, we circularized 5′- 32 P-labeled TER22 using T4 RNA ligase, as described previously [ 24 ] (Supplementary Fig. S3B, C).…”

Section: Resultsmentioning

confidence: 99%

RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity

Koralewska

Szczepańska

Ciechanowska

et al. 2021

Cell. Mol. Life Sci.

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Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform–PAZ–Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

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“…In contrast to in vitro transcription, chemical modifications can be easily incorporated into RNA chains during automated synthesis. If the oligomer is too long to be efficiently synthesized, two shorter modified RNA chains can be obtained separately and ligated using split-join ligation or T4 RNA ligase [72][73][74]. The other advantage of chemical synthesis is fewer constraints on the sequence of RNA and lower heterogeneity in comparison to in vitro transcription.…”

Section: Chemical Synthesismentioning

confidence: 99%

Overview of Methods for Large-Scale RNA Synthesis

et al. 2022

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In recent years, it has become clear that RNA molecules are involved in almost all vital cellular processes and pathogenesis of human disorders. The functional diversity of RNA comes from its structural richness. Although composed of only four nucleotides, RNA molecules present a plethora of secondary and tertiary structures critical for intra and intermolecular contacts with other RNAs and ligands (proteins, small metabolites, etc.). In order to fully understand RNA function it is necessary to define its spatial structure. Crystallography, nuclear magnetic resonance and cryogenic electron microscopy have demonstrated considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require large amounts of sample. Despite their limitations, chemical synthesis and in vitro transcription are usually employed to obtain milligram quantities of RNA for structural studies, delivering simple and effective methods for large-scale production of homogenous samples. The aim of this paper is to provide an overview of methods for large-scale RNA synthesis with emphasis on chemical synthesis and in vitro transcription. We also present our own results of testing the efficiency of these approaches in order to adapt the material acquisition strategy depending on the desired RNA construct.

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Stabilization of RNA hairpins using non-nucleotide linkers and circularization

Cited by 8 publications

References 45 publications

Circular L-RNA aptamer promotes target recognition and controls gene activity

Circular L-RNA aptamer promotes target recognition and controls gene activity

RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity

Overview of Methods for Large-Scale RNA Synthesis

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