Solution conformation of hexameric &amp; heptameric lariat-RNAs and their self-cleavage reactions which give products mimicking those from some catalytic RNAs (ribozymes)

Rousse, B.; Puri, Nitin; Viswanadham, G.; Agback, Peter; Glemarec, C.; Sandström, Anders; Sund, Christian; Chattopadhyaya, J.

doi:10.1016/s0040-4020(01)80852-8

Cited by 18 publications

(19 citation statements)

References 51 publications

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“…Gel electrophoresis equipment (APPENDIX 3B) with: 16 × 18-cm glass plates Spacers: 0.75 mm (analysis) or 1.5 mm (purification) Gel combs: 0.75 mm thick with 12 to 20 wells (analysis) or 1.5 mm thick with one to three large wells (purification) Sonicator or ∼50°C water bath (optional) 20 × 20-cm silica-coated thin-layer chromatography (TLC) plate with fluorescent indicator (e.g., Kieselgel 60 F 254 aluminum sheets) Handheld UV lamp (254 nm) Camera equipped with UV filter (optional) 90°C water bath or heating block (optional) UV shadow box Wrist-action shaker (optional) Speedvac evaporator (Savant) Sephadex G-25 (Amersham Pharmacia Biotech) or reversed-phase cartridges (e.g., Sep-Pak cartridges; Waters Chromatography) (1986) by solution-phase phosphoramidite chemistry. Chattopadhyaya and co-workers have focused on several strategies for the synthesis of small bRNA and RNA lariats with self-cleavage properties (Rousse et al, 1994). The authors' group has been describing in detail a methodology for the solid-phase synthesis of Y-shaped RNAs and DNAs, in which the branch point adenosine is linked to identical oligonucleotide chains via vicinal 2′,5′-and 3′,5′-phosphodiester linkages.…”

Section: Methodsmentioning

confidence: 99%

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Solid‐Phase Synthesis of Branched Oligonucleotides

Carriero

Damha

2002

CP Nucleic Acid Chemistry

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Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single-stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2',5'- and 3',5'-phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre-mRNA splicing. The protocols described herein allow for the facile solid-phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion-exchange HPLC and PAGE are also illustrated.

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Section: Methodsmentioning

confidence: 99%

“…Regiospecific synthesis of a tetranucleotide of natural branch point sequence was performed by Kierzek et al () by solution‐phase phosphoramidite chemistry. Chattopadhyaya and co‐workers have focused on several strategies for the synthesis of small bRNA and RNA lariats with self‐cleavage properties (Rousse et al, ).…”

Section: Commentarymentioning

confidence: 99%

Solid‐Phase Synthesis of Branched Oligonucleotides

Carriero

Damha

2002

CP Nucleic Acid Chemistry

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“…The strategy for the branch-point design in lariat-RNAs 6 and 7 is quite similar, but in these cases the protecting group strategies have been altered (see FIGS. 9 & 10, section 2.5. and 2.6. 2.2.…”

Section: Figmentioning

confidence: 99%

“…During this process, we have discovered that some of our synthetic models of lariat-RNAs undergo sitespecific self-cleavage in buffered aqueous solution at various temperatures and pH, in a concentration independent manner, to form an open chain product with 2',3'-cyclic phosphate and 5'-hydroxyl termini('-' ') (i.e. 8, 9 and 10 in FIG. 2), modelling the self-cleavage reaction of some catalytic RNAs (ribozymes).…”

Section: Introductionmentioning

confidence: 99%

The Synthesis of Lariat‐RNAs and their Conformational Analysis by NMR Spectroscopy: The Study of their Unique Self‐Cleavage Reaction Modelling Some Catalytic RNAs (Ribozymes)

Sund

Rousse

Puri

et al. 1994

Bulletin des Soc Chimique

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The synthesis of milligram quantities of lariat RNAs 1, 2, 5-7 and their cyclic analogs 3 and 4, modelling the lariat formed at the penultimate step of ligation of Group II and Nuclear mRNA introns in the Splicing reaction, have been achieved for the first time. These syntheses are highlighted by unique employment of various orthogonal protecting groups using phosphotriester and phosphoramidite chemistry. Some of the lariat-RNAs have been found to undergo site specific self-cleavage reaction to give an acyclic branched-RNA with 2',3'-cyclic phosphate and a 5'-hydroxyl termini, which is reminiscent of the products formed in some catalytic RI;JAs. These lariat-RNAs are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k = -1 min-at 37"C), and their rates of the self-cleavage is also much slower (k=0.25~10-~ min-' for lariat hexamer 5, and 0 . 1 6~1 0 -~ min-' for lariat heptamer 6 at 22°C). We have shown that the trinucleotidyl loop in the tetrameric 1 and pentameric 2 lariat-RNAs is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric 5 or heptameric 6 lariat-RNA does indeed have the required local and global conformation promoting the self-cleavage while the simple 2 ' 4 or 3'4-linked cyclic RNAs, 3 and 4, respectively, are completely stable and their structures are considerably different from the self-cleaving lariat-RNAs such as 5 or 6. The unique 3'-ethylphosphatefunction at the branch-point in 7, mimicking the 3'-tail of the lariat-hexamer 5, is the key structural feature that orchestrates its self-cleavage reaction (k =0.15~10-~ min" at 19°C) compared to the stable 2'+5'-linked cyclic RNA 3. The detailed conformational features of the self-cleaving lariat-RNAs 5, 6 and 7 by 500 MHz NMR spectroscopy and molecular dynamics simulations in the aqueous environment has been reviewed. A comparative study of the temperature dependence of the N o s equilibrium for the lariat tetrarner 7 and the 2'4-linked cyclic tetramer 3 shows that the A' residue in 7 is in 92% S-type conformation at 20°C whereas it is only in 55% S in 3 with a 3'-hydroxyl group. This displacement of the N o s pseudorotational equilibrium toward the S geometry is due to the enhanced gauche effect of the 3'-OP03Et' group at the branch-point adenosine in 7 compared to 3'-OH group in 3. This 3'-OP03Ef group promoted stabilisation of the S geometryat the branch-point by AH = 4 kcal.mol-' in 7 is contributing to the conformational driving force promoting its unique self-cleavage reaction. The comparison of AH" and AS" of the NoS pseudorotational equilibria in 7 and 3 clearly shows the remarkable effect of the 3'-ethylphosphate group in 7 in being able to dictate the conformational changes from the sugar moiety of the branch-point adenosine to the entire molecule (conformational transmission). Thus the S conformation in A', U2 and C6 sugar moieties is clearly thermodynamically more stabilised while it is considerably destabilised in G3 owing to the 3'-ethylphosphate group in 7 comqar...

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“…1,2 In nature, sugar moieties in DNA are present mainly in the South-type pseudorotamers which confer B-form global conformation, whereas the predominant North-type sugars in RNA renders it to a A-form global conformation. 3 It has been also found that some nucleotides are locked in to either South or North sugar conformation such as in ribozymes, 4 or lariat RNAs, 5,6 It is very likely that the conformation of the pentose-sugar moiety is a key factor 7 that determines the global conformation of nucleic acid, and consequently their physical, chemical and biological properties.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Structure of New Methylene-Bridged Hexopyranosyl Nucleoside (BHNA)

Zhou¹,

Plashkevych²,

Liu³

et al. 2009

HETEROCYCLES

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-A new member of hexopyranosyl nucleoside family, methylene-bridged hexopyranosyl nucleoside (BHNA), has been synthesized through generation of carbon radical at C6′ in [6′S-Me, 7′S-Me]-carba-LNA T nucleoside, followed by rearrangement to C4′ radical which was quenched by hydrogen atom to give BHNA. The stereoelectronic requirement for this unusual radical rearrangement has been elucidated by chemical model building and ab intio calculations to show that the coplanarity of the single electron occupied p-orbital at C6' with σ* O4'-C4' plays an important role for the rearrangement reaction to take place. The solution structure of BHNA has also been studied using NMR as well as by ab initio calculations. The new six-membered pyranosyl ring in BHNA, unlike other known hexopyranosyl nucleosides, adopts a twist conformation, with base moiety occupying the axial position while 3′-hydroxymethyl and 4′-hydroxyl occupying the equatorial position.

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Solution conformation of hexameric & heptameric lariat-RNAs and their self-cleavage reactions which give products mimicking those from some catalytic RNAs (ribozymes)

Cited by 18 publications

References 51 publications

Solid‐Phase Synthesis of Branched Oligonucleotides

Solid‐Phase Synthesis of Branched Oligonucleotides

The Synthesis of Lariat‐RNAs and their Conformational Analysis by NMR Spectroscopy: The Study of their Unique Self‐Cleavage Reaction Modelling Some Catalytic RNAs (Ribozymes)

Synthesis and Structure of New Methylene-Bridged Hexopyranosyl Nucleoside (BHNA)

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