Use of Deoxyribozymes in RNA Research

Silverman, Scott; Baum, Dana A.

doi:10.1016/s0076-6879(09)69005-4

Cited by 30 publications

(28 citation statements)

References 51 publications

(70 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We demonstrate that labeling yields can easily be determined upon site-specific cleavage into shorter fragments using the well-known RNAcleaving deoxyribozymes 8−17 or 10−23 (Figures 5c and S16). 16 Labeling of U6 snRNA at any targeted position between A40 and A56 was analyzed by cleavage of an analytical aliquot of each labeled RNA at nts 35 and 62, resulting in labeled 27-mers, which can easily be distinguished from the unlabeled 27-mer upon denaturing PAGE separation ( Figure 5d). Similarly, labeling between A62 and A94 was analyzed after cleavage at positions 51 and 97, resulting in labeled 46-mers which were separated and quantified (Figure 5e).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

2014

View full text Add to dashboard Cite

A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb 3+ as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2′-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2′,5′-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ∼160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA. ■ INTRODUCTIONThe site-specific attachment of labels onto RNA is of significant value in many areas of RNA chemical biology. The study of RNA folding and RNA−protein and RNA−ligand interactions by spectroscopic methods requires installation of observable reporter groups. Biochemical assays of RNA functions benefit from position-specific placement of reactive functional groups or probing agents. 1 Emissive nucleotide (nt) analogues or abiotic functional groups for bioorthogonal labeling can be sitespecifically installed by solid-phase synthesis. 2−4 Synthesis of RNAs longer than ∼40 nt often involves enzymatic manipulation or ligation using T4 ligases or deoxyribozymes. 5−9 As alternatives to the fragment-based approach, direct post-transcriptional labeling methods are gaining increasing attention. Recent developments include the use of specific methyltransferases together with functionalized Sadenosylmethionine (SAM) analogues, as exemplified by the alkylation of a specific guanosine in tRNA Phe with Trm1. 10 More generally applicable may be the reconstitution of C/D box small nucleolar ribonucleoprotein particles (snoRNPs), using guide RNAs responsible for the selection of the labeling site in the target RNA, as recently shown for the site-specific 2′-alkynylation of tRNA and mRNA. 11 A non-enzymatic example of oligonucleotide-guided post-transcriptional RNA modification was introduced with the functionality transfer reaction from 6-thioguanosine in a donor DNA to the exocyclic amino groups of cytosine and guanine residues in the target RNA. 12−14 Particularly attractive was the combination of a guide and an enzyme in a single strand of DNA, introduced as "DNA-catalyzed labeling of RNA". 15 In this approach, Baum and Silverman prepared a labeled tagging RNA by in vitro transcription using 5-aminoallyl-CTP and subsequent labeling by N-hydroxysuccinimide chemistry. This tagging RNA was then used as substrate for DNA-catalyzed ligation to the target RNA, forming labeled 2′,5′-branched RNA. Despite the elegance of t...

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

2014

View full text Add to dashboard Cite

show abstract

“…28 on site-specifically 32 P-labeled pre-mRNA. The pre-mRNA was produced by DNA splint-directed RNA ligation 74 of RNA fragments obtained by sitespecific DNA enzyme cleavage 75 of in vitro-transcribed actin pre-mRNA. Approximately 1 pmol of spliceosomes were eluted in 400 µl GK75 (75 mM KCl, 20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl 2 , 0.01% NP-40, and 5% glycerol) and subjected to UV cross-linking at a 254-nm wavelength for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Wysoczanski

Schneider

Munari

et al. 2014

Nat Struct Mol Biol

View full text Add to dashboard Cite

1 1 a r t i c l e sSplicing entails the removal of introns from pre-mRNAs to generate exon-only mRNA, which is exported out of the nucleus for translation 1 . The splicing process is driven and controlled by a large and dynamic RNA-protein complex, the spliceosome 1,2 . The composition and structure of the spliceosome undergoes multiple rearrangements during a splicing reaction, in which a set of distinct structural and compositional states, designated as E, A, B act , B* and C complexes, can be defined 1 . As part of this process, small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splice factors are recruited and released 1,2 . Although the organization of snRNP components of the spliceosome has received considerable attention in recent years, very little is known about the assembly, structure and dynamics of non-snRNP multimeric complexes.The non-snRNP RES complex is present in humans and yeast 3,4 . Deletion of RES genes slows splicing and leads to pre-mRNA leakage into the cytoplasm 3-5 . Distinct introns exhibit RES-dependent splicing 5-9 , as do pre-mRNAs encoding proteins functioning in RNA-nucleotide metabolism 8,10 . Components of the RES complex are found in B and C complexes of the spliceosome, in which RES can interact with U2 snRNP 4,11,12 . In yeast, the RES complex is composed of three proteins, snRNP-associated protein 17 (Snu17p, also known as Ist3p), pre-mRNA-leakage protein 1 (Pml1p) and bud siteselection protein 13 (Bud13p) 3,4 . Snu17p and Bud13p have been implicated directly in splicing 3,5,13 , whereas Pml1p has been linked to the retention of unspliced pre-mRNA in the nucleus 3,5 . Caenorhabditis elegans Bud13p is involved in embryogenesis 14 .Sequence analysis has indicated that Snu17p is a 148-residue (17.1-kDa) noncanonical member of the RRM family of proteins with a long C-terminal part, which exhibits low sequence similarity to published RRM structures 4 . Snu17p binds with nanomolar affinity to the 266-residue (30.5-kDa), natively disordered protein Bud13p 15 . The interaction has been postulated to involve a C-terminal UHM-ligand motif (ULM) in Bud13p that interacts with a U2AF-homology motif (UHM) in the RRM domain of Snu17p 13,15,16 . The only other identified domain encompasses a stretch of lysine residues at the N terminus of Bud13p. Binding of the third component, the 204-residue (23.4-kDa) Pml1p, occurs through its 50 N-terminal disordered residues. The remainder of Pml1p folds as a forkhead-associated domain 13,15,17 , which could potentially bind phosphopeptides 17 . Biochemical evidence has suggested that Snu17p acts as the central binding platform, which interacts with disordered parts of Bud13p and Pml1p 13,15,16 . The precise molecular architecture of the RES complex, however, has been elusive, and its RNA binding capabilities have remained unexplored.Here we solved the three-dimensional structure of the core of the RES complex and demonstrated that its assembly is driven by cooperativity that increases the binding affinity of the components of the complex ...

show abstract

“…This cleavage is often achieved with the additional feature of relatively broad RNA sequence generality, meaning that many different RNA substrate sequences may be cleaved merely by ensuring Watson-Crick complementarity between the RNA substrate and DNA enzyme. [20] In most cases, only a few particular RNA nucleotides near the cleavage site have restrictions on their sequence identities, and sets of deoxyribozymes have been developed that collectively allow practical cleavage of almost any RNA sequence. [20] …”

Section: Dna As a Catalystmentioning

confidence: 99%

DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol

Silverman

2010

Angew Chem Int Ed

Self Cite

231

View full text Add to dashboard Cite

DNA (deoxyribonucleic acid) is the genetic material common to all of Earth's organisms. Our biological understanding of DNA is extensive and well-exploited. In recent years, chemists have begun to develop DNA for nonbiological applications in catalysis, encoding, and stereochemical control. This Review summarizes key advances in these three exciting research areas, each of which takes advantage of a different subset of DNA's useful chemical properties.

show abstract

Use of Deoxyribozymes in RNA Research

Cited by 30 publications

References 51 publications

Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol

Contact Info

Product

Resources

About