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Current Protocols in Nucleic Acid Chemistry

Arindom Chatterjee, Chanchal K Malik, Ashis K Basu
This unit describes the detailed procedure in five parts for the synthesis of the C8-2'-deoxyguanosine-3-aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2'-deoxyguanosine, O(6) -benzyl-N(2) -DMTr-3'-5'-bisTBDMS-C8-Br-2'-deoxyguanosine, is described in the first part. The synthesis of the reduced carcinogen 3-aminobenzanthrone is detailed in part two. The third part outlines the key step of the adduct formation between the reduced carcinogen and the protected nucleoside by a palladium-catalyzed cross coupling reaction...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Geeta Meher, Nabin K Meher, Radhakrishnan P Iyer
Oligonucleotides carrying a variety of chemical modifications including conjugates are finding increasing applications in therapeutics, diagnostics, functional genomics, proteomics, and as research tools in chemical and molecular biology. The successful synthesis of oligonucleotides primarily depends on the use of appropriately protected nucleoside building blocks including the exocyclic amino groups of the nucleobases, the hydroxyl groups at the 2'-, 3'-, and 5'-positions of the sugar moieties, and the internucleotide phospho-linkage...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Oleksandr Plashkevych, Ram Shankar Upadhayaya, Jyoti Chattopadhyaya
In light of the impressive gene-silencing properties of carba-LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof-of-concept for biochemical applications in post-transcriptional gene silencing, we envision the true potential of carba-LNA modifications to be revealed soon. Herein we provide detailed protocols for synthesis of carba-LNA-A, -G, -(5-Me) C, and -T nucleosides on a medium/large scale (gram scale), as well as important guidelines for incorporation of these modified carba-LNAs into DNA or RNA oligonucleotides...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Ali Nikoomanzar, Matthew R Dunn, John C Chaput
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Anaïs Depaix, Suzanne Peyrottes, Béatrice Roy
This unit describes a one-pot, two step synthesis of ribonucleoside 5'-di- and 5'-triphosphates, as well as their purification. The first step of the synthesis involves the activation of an unprotected ribonucleoside 5'-monophosphate with 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate and imidazole, in a mixture of water/acetonitrile. The resulting phosphorimidazolate intermediate is then treated with inorganic phosphate or pyrophosphate to afford the corresponding nucleoside 5'-di- or 5'-triphosphates...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Pramod K Sahu, Dnyandev B Jarhad, Gyudong Kim, Lak Shin Jeong
5'-Homo-4'-selenonucleosides, a class of next-generation nucleosides, are synthesized from D-ribose via a 4-selenosugar intermediate. The key step in synthesizing this intermediate is a seleno-Michael reaction. 5'-Homo-4'-selenouridine and -adenosine are prepared using Pummerer-type and Vorbrüggen condensation, respectively. © 2017 by John Wiley & Sons, Inc.
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Andrzej Grajkowski, Jacek Cieślak, Serge L Beaucage
An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
Jon D Moulton
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Jolanta Brzezinska, Agnieszka Witkowska, Tomasz P Kaczyński, Dominika Krygier, Tomasz Ratajczak, Marcin K Chmielewski
Application of 2-pyridinyl thermolabile protecting groups (2-PyTPGs) for protection of hydroxyl, phosphate, and carboxyl functions is presented in this unit. Their characteristic feature is a unique removal process following the intramolecular cyclization mechanism and induced only by temperature rise. Deprotection rate of 2-PyTPGs is dependent on certain parameters, such as solvent (aqueous or non-aqueous medium), pH values, and electron distribution in a pyridine ring. The presented approach pertains not only to protecting groups but also to an advanced system of controlling certain properties of 2-pyridinyl derivatives...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Rui Wang, Phensinee Haruehanroengra, Jia Sheng
This unit describes the chemical synthesis of the S-geranyl-2-thiouridine (ges(2) U) phosphoramidite and its incorporation into RNA oligonucleotides through solid-phase synthesis. Starting from the 2-thiouracil nucleobase and the protected ribose, the 2-thiouridine is synthesized and the geranyl functionality is introduced into the 2-thio position by using geranyl bromide as the geranylating reagent before the conversion of this modified nucleoside into a phosphoramidite building block. The modified phosphoramidite is used to make the geranyl-RNA oligonucleotides with a solid-phase DNA synthesizer...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Rafael Del Villar-Guerra, Robert D Gray, Jonathan B Chaires
Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Jyotirmoy Maity, Smriti Srivastava, Yogesh S Sanghvi, Ashok K Prasad, Roger Stromberg
Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5-Bromouridine, 5-bromo-2'-deoxyuridine, 5-bromouridine-5'-triphosphate, and nucleotides containing 5-bromouridine have been tested and used for numerous biological studies. 8-Bromopurine nucleosides have been used as essential precursors for the synthesis of nucleosides with fluorescent properties. This unit describes protocols for the synthesis of bromonucleosides using sodium monobromoisocyanurate (SMBI) in a straightforward way...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Yoshiaki Masaki, Mitsuo Sekine, Kohji Seio
Chemical modification of RNA duplexes alters their stability. We have attempted to develop a computational approach to estimate the thermal stability of chemically modified duplexes. These studies revealed that the deformability of chemically modified RNA duplexes, calculated from molecular dynamics simulations, could be used as a good indicator for estimating the effect of chemical modification on duplex thermal stability. This unit describes how deformability calculation can be applied to estimate the relative stability of chemically modified RNA duplexes...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
Muthian Shanmugasundaram, Annamalai Senthilvelan, Anilkumar R Kore
This unit delineates a simple, reliable, straight-forward, general, and efficient chemical method for the synthesis of modified nucleoside-5'-O-triphosphates such as 5-methylcytidine-5'-O-triphosphate (5-Me-CTP), pseudouridine-5'-O-triphosphate (pseudo-UTP), and N(1) -methylpseudouridine-5'-O-triphosphate (N(1) -methylpseudo-UTP), starting from the corresponding nucleoside. The reaction utilizes an improved protection-free "one-pot, three-step" Ludwig synthetic strategy that involves the monophosphorylation of the nucleoside with phosphorous oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding ribonucleoside triphosphate (NTP) in moderate yields...
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
Jiali Yang, Lijia Yu, Liangliang Zhang, Xingsu Long, Yuzhuo Ji, Xinjing Tang
RNA-induced gene silencing has been widely applied as a powerful research tool in drug development due to its sequence-specific degradation of target mRNA. Conditional regulation of gene functions with small interfering RNAs (siRNAs) is highly useful, especially when specific gene expression regulation with spatiotemporal resolution and amplitude is desired. Here, the synthesis of a series of new caged siRNAs with vitamin E (vitE) modification and/or a single photolabile linker at the 5' terminal is described...
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
Xinhui Lou, Martin Egli, Xianbin Yang
Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol)...
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
David H Mathews, Douglas H Turner, Richard M Watson
In this unit, protocols are provided for predicting RNA secondary structure with the user-friendly RNAstructure desktop computer program and the RNAstructure Web server. The minimum free energy structure and a set of suboptimal structures with similar free energies are predicted. Prediction of high-affinity oligonucleotide binding sites to a structured RNA target is also presented. © 2016 by John Wiley & Sons, Inc.
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
Marcel Hollenstein, Masad J Damha
Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility with isothermal amplification strategies. In this context, rolling circle amplification (RCA) is a wide-spread enzymatic replication method in which small single-stranded DNA (ssDNA) circles serve as templates in primer extension reactions yielding very long, ssDNA products...
December 1, 2016: Current Protocols in Nucleic Acid Chemistry
Jingshu Guo, Robert J Turesky
Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the etiological role of a genotoxic chemical in cancer risk. The analytical chemist is confronted with a great challenge because the levels of DNA adducts generally occur at <1 adduct per 10(7) nucleotides, and the amount of tissue available for measurement is limited...
September 1, 2016: Current Protocols in Nucleic Acid Chemistry
Derek K O'Flaherty, Christopher J Wilds
This unit describes the preparation O(6) -2'-deoxyguanosine-butylene-O(6) -2'-deoxyguanosine dimer phosphoramidites and precursors for incorporation of site-specific intrastrand cross-links (IaCL) into DNA oligonucleotides. Protected 2'-deoxyguanosine dimers are produced using the Mitsunobu reaction. IaCL DNA containing the intradimer phosphodiester are first chemically phosphorylated, followed by a ring-closing reaction using the condensing reagent 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole. Phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis and standard deprotection and cleavage protocols are employed...
September 1, 2016: Current Protocols in Nucleic Acid Chemistry
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