An Efficient Method for the Isolation and Purification of Oligoribonucleotides

Sproat, Brian S.; Colonna, Francesco Paolo; Mullah, Bashar; Tsou, Dean; Andrus, Alex; Hampel, Arnold; Vinayak, Ravi

doi:10.1080/15257779508014668

Cited by 132 publications

(94 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…CD Spectra-For CD spectra, R18, D18, and chimeric DNA-RNA oligonucleotides were gel-purified on denaturing 16% polyacrylamide-TBE gels, eluted, and desalted as described (63,64). To determine the concentration of the synthetic oligonucleotides, an extinction coefficient (⑀ 260 ) of 14.3 ϫ 10 5 M Ϫ1 cm Ϫ1 was used.…”

Section: Methodsmentioning

confidence: 99%

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Iwatani¹,

Rosen²,

Guo³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Iwatani¹,

Rosen²,

Guo³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Puri®cation of the oligoribonucleotides was carried out by anion exchange chromatography on a NucleoPac PA-100 column (Dionex) (Sproat et al, 1995;Schmidt et al, 1996b) using buffer A: 10 mM and buffer B: 400 mM sodium perchlorate, 20 mM Tris-HCl (pH 6.8), 25% formamide,¯ow rate 3 ml min À1 with gradients of 15 to 45% B over 20 minutes. Desalting was achieved via extensive dialysis against water.…”

Section: Methodsmentioning

confidence: 99%

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme

Earnshaw

Masquida

Müller

et al. 1997

Journal of Molecular Biology

View full text Add to dashboard Cite

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2 H -2 H disulphide linkages of known spacing (12 or 16 A Ê ) between de®ned ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5 H -GAR-3 H -motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a``ribose zipper'', another group I ribozyme feature, formed between the hydroxyl groups of residues A 10, G 11 of domain A and C 25 , A 24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the speci®c phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

show abstract

“…A solution of 0.5 M iodine in pyridine is the standard reagent used in DNA synthesis to oxidize the phosphite to the phosphate. In our synthesis of RNA, we replaced this with a solution of 2 M tert-butyl hydroperoxide (TBHP) in toluene/dichloromethane (27) to avoid exposure of the vinyl moiety to pyridine. We also used 3% dichloroacetic acid in dichloromethane, a mild acid, to effect the removal of the 5'-dimethoxytrityl groups.…”

Section: Resultsmentioning

confidence: 99%

“…As described earlier, the λ em was 378 nm. The quantum yield, ϕ, was determined to be 0.37 relative to tryptophan (ϕ = 0.14) (45) and 0.33 relative to 2-aminopurine (ϕ = 0.66) (27). These determinations were made on absorbance matched samples in water at pH 7.0 and 25°C.The extended conjugation of 8-vinyladenine makes it a better leaving group than adenine.…”

Section: -Vinyladenine: Synthesis and Physicochemical Characterizationmentioning

confidence: 99%

Vinyldeoxyadenosine in a Sarcin−Ricin RNA Loop and Its Binding to Ricin Toxin A-Chain

2007

View full text Add to dashboard Cite

Abstract8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure to characterize the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome inactivating proteins. Ricin Toxin A-chain (RTA) and Pokeweed Antiviral Protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze 8-vinyladenine release from 8vdA-10. Molecular dynamics simulations implicate a role for Arg 180 in oxacarbenium ion destabilization and lack of catalysis. However, 8vdA-10 is an active site analog and inhibits RTA with a K i value of 2.4 μM. Adenine is also released from the second adenosine in the modified tetraloop demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analog defines the specificities of RTA for the two adenylate depurination sites in an RNA substrate with a GAGA tetraloop. The rate of non-enzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

show abstract

An Efficient Method for the Isolation and Purification of Oligoribonucleotides

Cited by 132 publications

References 39 publications

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme

Vinyldeoxyadenosine in a Sarcin−Ricin RNA Loop and Its Binding to Ricin Toxin A-Chain

Contact Info

Product

Resources

About