Chemical synthesis of branched RNA

Kierzek, Ryszard; Kopp, David W.; Edmonds, Mary; Caruthers, Marvin H.

doi:10.1093/nar/14.12.4751

Cited by 56 publications

(27 citation statements)

References 22 publications

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“…The synthesis began by assembly of the linear tridecanucleotide 5′-d(TACTA)-rA-d(CAA GTT)-3′ in the normal 3′-to-5′ fashion. Following acetylation of the terminal 5′-hydroxyl group, the support was treated with triethylamine/acetonitrile solution (4:6 v/v, 1.5 h, room temperature) to affect the removal of all cyanoethyl phosphate protecting groups, thus providing an oligomer with a intrinsically more stable phosphodiester backbone (25). This is necessary because deblocking of a 2′-OH group vicinal to a 3′,5′-phosphotriester linkage under neutral, acidic, or basic conditions has been shown to lead to phosphoryl migration and/or chain cleavage (26)(27)(28)(29).…”

Section: Synthesis Of Branched Oligodeoxynucleotidesmentioning

confidence: 99%

Regiospecific Solid-Phase Synthesis of Branched Oligonucleotides. Effect of Vicinal 2‘,5‘- (or 2‘,3‘-) and 3‘,5‘-Phosphodiester Linkages on the Formation of Hairpin DNA

Braich

Damha

1997

Bioconjugate Chem.

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A general procedure for the solid-phase regiospecific synthesis of branched oligonucleotides (bNA) analogues using readily available phosphoramidite reagents has been developed. The key feature of this method is use of the solid-phase phosphoramidite procedure to assemble linear oligonucleotide sequences and sequential removal of the phosphate (beta-cyanoethyl or methyl) and silyl protecting groups without detaching the nascent oligonucleotide from the solid support. Conversion of the phosphate backbone into the more stable phosphodiester linkages allows for removal of the 2'-O-tert-butyldimethylsilyl protecting group without cleavage or isomerization at the branch point. This method allows for the formation of branched oligonucleotides with sequences of arbitrary base composition, length, and orientation around the branch point junction, including a "Y"-shaped octadecamer d(TACTA)-rA[2',5'd(GTATGT)]3',5'd(CAAGTT). Studies to explore structural effects in the use of a branched adenosine as replacement for nucleotide loops in duplex and triplex DNA are also described. Branched oligonucleotides of the type rA[2',5'dCndA10-5']3',5'dCndT10-3' and rA[2',5'dCn3',3'dA10-5']3',5'dCnT10-3' form hairpin duplexes with thermal stability comparable to or better than that of one with a natural deoxynucleotide loop.

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Section: Synthesis Of Branched Oligodeoxynucleotidesmentioning

confidence: 99%

Regiospecific Solid-Phase Synthesis of Branched Oligonucleotides. Effect of Vicinal 2‘,5‘- (or 2‘,3‘-) and 3‘,5‘-Phosphodiester Linkages on the Formation of Hairpin DNA

Braich

Damha

1997

Bioconjugate Chem.

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“…38 The synthesis of 5´ O (4,4´ dimethoxytrityl) derivative 7 followed by its 3´ phosphitylation was carried out according to standard procedures. 39 The phosphitylation time was increased to 2 h, which was associated with steric hindrance of the 3´ hydroxy group in compound 7.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and properties of oligodeoxyribonucleotides containing 2’-O-(2,3-dihydroxypropyl)- and 2’-O-(2-oxoethyl)arabinouridine residues

et al. 2005

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2´ O (2,3 Dihydroxypropyl)arabinouridine containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2´ O (2,3 dihydroxypropyl)arabino uridine, and the corresponding 3´ phosphoramidite. Oxidation of these oligodeoxyribo nucleotides with sodium periodate afforded oligonucleotides containing 2´ O (2 oxo ethyl)arabinouridine residues. Subsequent modification of the aldehyde containing oligonucle otides involved the reactions with 9 hydrazinoacridine and N aminooxyacetyl peptide and reductive amination by 4 (1 pyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyribo nucleotides are slightly lower than those of natural duplexes. Duplexes with complementary oligoribonucleotides are substantially destabilized.Oligonucleotides including modified oligonucleotides find wide application in various fields of molecular biol ogy. They are used as primers for DNA sequencing and amplification, for the determination of DNA primary structures by hybridization, and also as potential thera peutics. 2,3 In particular, modified oligonucleotides are employed for the preparation of conjugates with various compounds, such as peptides, oligosaccharides, and fluo rescent dyes for studying the behavior of nucleic acids in vitro or in vivo. Affinity modification of DNA binding proteins is used to study the structures and functioning of the active sites of the latter. 4,5Conjugation of fluorophores or electrochemically ac tive compounds to oligonucleotides enables the use of such derivatives in the design of DNA detection systems by hybridization. 6-8 Marker groups substantially increase sensitivity of DNA detection. Many conjugates of oligo nucleotides with peptides, carbohydrates, and lipophilic compounds more easily penetrate cell membranes than native nucleic acids, and this fact can be used in antisense and antigene biotechnology. 9-11Covalent attachment of proteins to nucleic acids is the method of choice for solving a series of problems. First and foremost, it allows identification of the amino acids interacting with particular regions of nucleic acids in nucleic acid-protein complexes. In studies of multi protein complexes formed during the replication, transla tion, etc., one can determine a protein component inter acting with a region of nucleic acid recognition 12 and examine cell extracts with the aim of revealing proteins that selectively interact with particular nucleotide se quences. This approach is also applicable in the SELEX technology for the design of nucleic acid aptamers that irreversibly bind to proteins. 13,14 In recent years, consid erable progress has been achieved in crystallization of covalent nucleic acid-protein complexes and elucida tion of their structures by X ray diffraction. 15 Earlier, we have demonstrated that 2´ O (2 oxo ethyl)uridine containing DNA duplexes can successfully be used for the affinity modification of DNA binding proteins, such as transcription factor NF κB 1...

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“…The standard 2',3' ADP was produced by treating the tetraribonucleotide branch pGA4 (i.e. adenosine branched 2',5' to G and 3',5' to C) (Kierzek et al, 1986; a gift of Dr Mary Edmonds, Unviersity of Pittsburgh) with RNase TI, then performing a NaIO4 oxidation and (3-elimination (see below) to leave 2',3' ADP. Triethylamine-bicarbonate (TEAB) was made by bubbling CO2 (from dry ice) through a 1 M solution of redistilled triethylamine until the pH reached -7.0.…”

Section: Methodsmentioning

confidence: 99%

Trypanosome trans-splicing utilizes 2′-5′ branches and a corresponding debranching activity.

Sutton

Boothroyd

1988

The EMBO Journal

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The 5′ ends of trypanosome mRNAs consist of an identical sequence of 39 nucleotides which is derived from a discrete transcript of approximately 140 nucleotides (medRNA). It has been proposed that generation of chimeric mRNAs in trypanosomes occurs by the process of trans‐splicing involving medRNA and an acceptor RNA. Part of the basis for this suggestion comes from the ability of HeLa cell extracts (known to contain debranching activity) to catalyze the release of the intron portion of medRNA (minRNA) implying a Y‐branched intermediate in the splicing process. Here we provide direct chemical analysis that miniRNA is attached to higher mol. wt RNA molecules by a 2′‐5′ phosphodiester bond (i.e. as a branched structure). We also demonstrate that trypanosomes have substantial amounts of debranching activity which is similar in nature to that of HeLa cells. These results provide further evidence for trans‐splicing in trypanosomes and highlights its similarity to cis‐splicing in other eukaryotes.

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Chemical synthesis of branched RNA

Abstract: A branched tetranucleotide consisting of adenosine linked 2' and 5' to guanosine and 3' to cytidine was synthesized from appropriately protected nucleoside phosphoramidites as synthons. The product was characterized enzymatically.

Cited by 56 publications

References 22 publications

Regiospecific Solid-Phase Synthesis of Branched Oligonucleotides. Effect of Vicinal 2‘,5‘- (or 2‘,3‘-) and 3‘,5‘-Phosphodiester Linkages on the Formation of Hairpin DNA

Regiospecific Solid-Phase Synthesis of Branched Oligonucleotides. Effect of Vicinal 2‘,5‘- (or 2‘,3‘-) and 3‘,5‘-Phosphodiester Linkages on the Formation of Hairpin DNA

Synthesis and properties of oligodeoxyribonucleotides containing 2’-O-(2,3-dihydroxypropyl)- and 2’-O-(2-oxoethyl)arabinouridine residues

Trypanosome trans-splicing utilizes 2′-5′ branches and a corresponding debranching activity.

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