A general two-step strategy to synthesize lariat RNAs

Wang, Yangming; Silverman, Scott

doi:10.1261/rna.2259406

Cited by 12 publications

(14 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This will be of high significance for selective and internal nucleic acid labeling with multiple dyes on the same RNA, as for example requested by multicolor single-molecule FRET techniques 46 or for nucleic acid cyclization, 47 lariat formation or branching. 48 The possibility of reverse attachments will be of further interest in the emerging field of RNA nanotechnologies. 49 , 50 …”

Section: Resultsmentioning

confidence: 99%

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Fauster¹,

Hartl²,

Santner³

et al. 2012

ACS Chem. Biol.

111

116

View full text Add to dashboard Cite

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

show abstract

Section: Resultsmentioning

confidence: 99%

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Fauster¹,

Hartl²,

Santner³

et al. 2012

ACS Chem. Biol.

111

116

View full text Add to dashboard Cite

show abstract

“…Independent of the chemical or enzymatic preparation of the linear precursor, the 5′-terminus needs to be phosphorylated to provide the functionality required for ligation. Phosphorylation can be performed chemically or enzymatically ( 71 ) followed directly by the circularization reaction ( 72 ) (Supplementary data, Protocols S3 and S4). In the case of enzymatically prepared RNA substrates, the 5′-terminal triphosphate resulting from the in vitro transcription reaction must be removed prior to phosphorylation by a polynucleotide kinase.…”

Section: Circularization In Vitromentioning

confidence: 99%

RNA circularization strategies in vivo and in vitro

Petkovic

Müller

2015

Nucleic Acids Research

278

218

View full text Add to dashboard Cite

In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.

show abstract

“…Separately, one of the deoxyribozymes found during development of the 8–17 selection pressure (6CE8)11 was shown to be useful for creating branched RNA with any branch-site nucleotide,16 which is one of the only nucleotides whose identity is restricted for 7S11. We also showed that another deoxyribozyme (6BX22) can be used for one-step synthesis of lariat RNAs directly related to biological sequences,17 and we developed a two-step ligation route to lariats that cannot be made efficiently in one step 18…”

Section: Rna Ligation With 5′-triphosphate Substratesmentioning

confidence: 99%

Deoxyribozymes: Selection Design and Serendipity in the Development of DNA Catalysts

2009

Self Cite

View full text Add to dashboard Cite

CONSPECTUSOne of the chemist's key motivations is to explore the forefront of catalysis. In this Account, we describe our laboratory's efforts at one such forefront: the use of DNA as a catalyst.Natural biological catalysts include both protein enzymes and RNA enzymes (ribozymes), whereas nature apparently uses DNA solely for genetic information storage. Nevertheless, the chemical similarities between RNA and DNA naturally lead to laboratory examination of DNA as a catalyst, especially because DNA is more stable than RNA and is less costly and easier to synthesize. Many catalytically active DNA sequences (deoxyribozymes, also called DNAzymes) have been identified in the laboratory by in vitro selection, in which many random DNA sequences are evaluated in parallel to find those rare sequences that have a desired functional ability. Since 2001, our research group has pursued new deoxyribozymes for various chemical reactions. We consider DNA simply as a large biopolymer that can adopt intricate three-dimensional structure and, in the presence of appropriate metal ions, generate the chemical complexity required to achieve catalysis.Our initial efforts focused on deoxyribozymes that ligate two RNA substrates. In these studies, we used only substrates that are readily obtained biochemically. Highly active deoxyribozymes have been identified, with emergent questions regarding chemical selectivity during RNA phosphodiester bond formation. Deoxyribozymes allow synthesis of interesting RNA products, such as branches and lariats, that are otherwise challenging to prepare. Our experiments have demonstrated that deoxyribozymes can have very high rate enhancements and chemical selectivities. We have also shown how the in vitro selection process itself can be directed towards desired goals, such as selective formation of native 3′-5′ RNA linkages. A final lesson is that unanticipated selection outcomes can be very interesting, highlighting the importance of allowing such opportunities in future experiments.More recently, we have begun using non-oligonucleotide substrates in our efforts with deoxyribozymes. We have especially focused on developing DNA catalysts for reactions of small molecules or amino acid side chains. For example, new deoxyribozymes have the catalytic power to create a nucleopeptide linkage between a tyrosine or serine side chain and the 5′-terminus of an RNA strand. Although considerable further work remains to establish DNA as a practical catalyst for small molecules and full-length proteins, the progress to date is very promising. The many lessons learned during the experiments described in this Account will help us and others to realize the full catalytic power of DNA. † This paper is dedicated to the memory of Christopher Foote, my undergraduate mentor at UCLA and senior editor of this journal from 1995 until his death in 2005. *scott@scs.illinois.edu . Why Pursue DNA as an Artificial Catalyst? NIH Public AccessOf the three natural biopolymers DNA, RNA, and proteins, only the last two are known to have e...

show abstract

A general two-step strategy to synthesize lariat RNAs

Cited by 12 publications

References 48 publications

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

RNA circularization strategies in vivo and in vitro

Deoxyribozymes: Selection Design and Serendipity in the Development of DNA Catalysts

Contact Info

Product

Resources

About