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RECENTLY PUBLISHED:
Unit 4.14 Solid-Phase Synthesis of Branched Oligonucleotides (S. Carriero and M.J. Damha, McGill University). 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 specificify 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.
Unit 11.8 Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways (P.C. Bevilacqua, Pennsylvania State University; D.H. Turner, University of Rochester). This unit discusses fluorescence spectroscopy as a tool for studying RNA folding. Ribozymes and oligonucleotides can be labeled with a fluorescent probe and analyzed to give information about both slow and fast kinetic processes with real-time data acquisition. The unit discusses the advantages and disadvantages of various pendant probes and nucleotide analogs, the analytical methods that can be used, instrument setup, control experiments, and a variety of kinetic experiments that can be performed, such as determination of rate constants.
Unit 11.9 Use of Chemical Modification to Elucidate RNA Folding Pathways (D.H. Mathews and D.H. Turner, University of Rochester). Chemical modification is sensitive to the accessibility of a nucleotide to the solvent, and many nucleotides become less accessible as an RNA folds into its structured form. Chemical modification reagents are therefore suitable for following RNA folding, and can be used to study the kinetics of structure formation on time scales ranging from minutes to hours.
New chapter on DNA nanotechnology!
Unit 12.1 Key Experimental Approaches in DNA Nanotechnology (N.C. Seeman, New York University). DNA nanotechnology combines unusual DNA motifs with sticky-ended cohesion to build polyhedral objects, topological targets, nanomechanical devices, and both crystalline and aperiodic arrays. The goal of DNA nanotechnology is control of the structure of macroscopic matter on the finest possible scale. Applications are expected to come in the areas of X-ray crystallography, nanoelectronics, nanorobotics, and DNA-based computation. DNA and its close molecular relatives appear extremely well suited for these goals. This discussion includes the generation of new DNA motifs, construction methods (synthesis, hybridization, phosphorylation, ligation), and a variety of methods for characterization of motifs, devices, and arrays. Finally, the use of DNA nanotechnology as a tool in biochemistry is discussed.
Unit 12.2 Preparation of Gold Nanoparticle-DNA Conjugates (T.A. Taton, University of Minnesota). This unit describes the preparation of conjugates between nanometer-scale gold particles and synthetic oligonucleotides. Oligonucleotide-functionalized gold nanoparticles are finding increased use in both the construction of complex, tailored nanostructures and the optimization of DNA sequence analysis. The protocols in this unit outline the synthesis, purification, and characterization of nanoparticle-DNA conjugates for applications in nanotechnology and biotechnology. Separate procedures are presented for nanoparticles functionalized with just one or a few oligonucleotide strands and for nanoparticles functionalized with a dense layer of oligonucleotide strands. The different physical and chemical properties of these two types of conjugates are discussed, as are their stability and utility in different environments.
FORTHCOMING:
Unit 1.5 Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code (F.E. Romesberg, C. Yu, S. Matsuda, and A.A. Henry, The Scripps Research Institute). Previous efforts to generate orthogonal base pairs have relied on hydrogen bonding patterns that are not found in the canonical Watson-Crick base pairs. An alternative strategy involves the development of unnatural bases that form pairs by interbase hydrophobic interactions. Such hydrophobic bases should not pair stably opposite natural bases due to the forced desolvation of the purines or pyrimidines. This unit describes the design, synthesis, and purification of unnatural 1-b-d-2-deoxyribosyl-N-nucleosides and C-nucleosides. To be useful in extending the genetic code, unnatural nucleosides must form stable and selective base pairs and must be good substrates for DNA polymerases. Protocols are thus presented for determining thermodynamic and kinetic properties of unnatural nucleosides.
Unit 1.6 Syntheses of 15N-Specifically Labeled Adenosine and Guanosine (B.L. Gaffney and R.A. Jones, Rutgers University). 15N-Specifically labeled nucleosides are primarily used for NMR, but can also be used for mass spectrometry because of their additional mass. This unit describes the specific incorporation of 15N into the N7 and amino positions of adenosine, and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions. Furthermore, variations of the procedures are presented that include 13C at C8 of adenosine and C8 or C2 of guanosine. These 13C tags permit incorporation of two 15N-labeled nucleosides into an RNA strand while ensuring that their NMR signals can be distinguished from each other by the presence or absence of C-N coupling.
Unit 1.7 Synthesis of Protected 2¢ -Deoxy-2¢ -fluoro-b -d-arabinonucleosides (M.I. Elzagheid, E. Viazovkina, and M.J. Damha, McGill University). A number of highly selective nucleoside antiviral and antileukemic agents have been based on 2-deoxy-2-fluoroarabinose. These compounds also serve as building blocks for 2¢ -deoxy-2¢ -fluoroarabinonucleic acids, which form a more stable duplex with RNA than a DNA:RNA duplex of identical sequence. Such duplexes are substrates for RNase H, which is involved in the mechanism of action of antisense oligonucleotides. This unit presents the synthesis, purification, and characterization of the four 2¢ -deoxy-2¢ -fluoroarabinonucleosides. Synthesis of phosphoramidites and oligonucleotides using these nucleosides is presented in Unit 4.15.
Unit 3.9 The 3-[(N-tert-Butyl)carboxamido]-1-propyl and 4-Oxopentyl Groups for Phosphate/Thiophosphate Protection in the Synthesis of Oligodeoxyribonucleotides (A. Wilk, M.K. Chmielewski, A. Grajkowski, and S.L. Beaucage, Food and Drug Administration; L.R. Phillips, National Cancer Institute). The detailed preparation of deoxyribonucleoside phosphoramidites bearing a 3-[(N-tert-butyl)carboxamido]-1-propyl or 4-oxopentyl group for P(III) protection is presented. The use of these groups circumvents nucleobase alkylation during oligonucleotide deprotection and offers flexibility in the choice of deprotection conditions. Synthesis of the appropriate phosphoramidites is conveniently accomplished in a one-pot reaction by mixing phosphorus trichloride and N,N-diisopropylamine, and then adding either (N-tert-butyl)-4-hydroxybutyramide or 3-acetyl-1-propanol. The phosphinylation of suitably protected deoxyribonucleosides is also described.
Unit 4.15
Solid-Phase Synthesis of 2¢ -Deoxy-2¢ -fluoro-b -d-arabino Oligonucleotides
and Their Phosphorothioate Derivatives (E. Vaizovkina, M. Mangos, M.I. Elzagheid,
and M.J. Damha, McGill University). This unit describes the solid-phase synthesis
oligofluoroarabinonucleotides that contain phosphodiester (PO) or phosphorothioate
(PS) internucleotide linkages. Protocols include preparation of phosphoramidites
from 2¢ -deoxy-2¢ -fluoroarabinonucleosides (Unit 1.7), assembly
on a DNA synthesizer, and final deprotection and purification. The standard
coupling cycles used for DNA synthesis have been optimized, and also take
monomer concentrations into consideration. Additionally, methods for detecting
undesired oxidation in PS sequences are presented that enable a critical survey
of various sulfurization conditions used to enhance the fidelity of PS incorporation.
