bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemistry of CpG DNA
Authors: Kandimalla Ekambar R. , Agrawal Sudhir 
Published: June 2004

AbstractThe vertebrate immune system can recognize specific pathogen‐associated molecular patterns in invading microorganisms, including the unmethylated CpG dinucleotide. This unit discusses the receptors that recognize CpG motifs and important aspects of the sequence context of CpG motifs to the end of understanding and designing CpG DNA for therapeutic purposes.
Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages
Authors: Guga Piotr , Stec Wojciech J. 
Published: June 2004

AbstractA method for solid‐phase synthesis of stereodefined PS‐oligos via an oxathiaphospholane approach using pure P‐diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5′‐O‐DMTr‐N‐protected deoxyribonucleosides with 2‐chloro‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O‐labeled mercaptoalcohol, the corresponding 18O‐labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS‐oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s into their P‐diastereomers, and for manual solid‐phase synthesis of PS‐oligos. Oxidation of 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s with selenium dioxide yields their 2‐oxo‐analogs, which are suitable either for elongation of stereodefined PS‐oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O‐labeled PO‐oligos of predetermined P‐chirality.
Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification
Authors: Fang Shiyue , Bergstrom Donald E. 
Published: June 2004

AbstractThe preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5′‐terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5′‐OH and 5′‐phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin‐coated microspheres, full‐length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high‐quality, full‐length, unmodified 5′‐OH and 5′‐phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.
Stepwise Solid‐Phase Synthesis of Nucleopeptides
Authors: Grandas Anna , Marchán Vicente , Debéthune Laurent , Pedroso Enrique 
Published: June 2004

AbstractPhosphodiester‐linked peptide‐oligonucleotide conjugates (nucleopeptides) are obtained by stepwise solid‐phase procedures. The peptide is first assembled on a suitably derivatized solid matrix and the oligonucleotide is subsequently elongated at the free hydroxyl group of the linking amino acid. Temporary acid‐labile and permanent base‐labile protecting groups are combined. Careful choice of the protection scheme is required to prevent and minimize side reactions that may degrade the target molecule.
Chemical and Enzymatic Methods for Preparing Circular Single‐Stranded DNAs
Authors: Diegelman Amy M. , Kool Eric T. 
Published: June 2004

AbstractSmall circular oligonucleotides can be used for diagnostic, therapeutic, and laboratory purposes. These systems have gained considerable attention in recent years because they form unusually strong and specific complexes with RNA and DNA strands. Synthetic circular DNAs of 20 to 200 nucleotides can also serve as catalysts for amplified DNA and RNA synthesis by a rolling circle mechanism. This unit presents methods for synthesizing small circular oligonucleotides. These simple “one‐pot” procedures are carried out using short DNA splints that hold the circle together until it is chemically or enzymatically ligated.
Synthesis of Endcap Dimethoxytrityl Phosphoramidites for Endcapped Oligonucleotides
Authors: Pingle Maneesh R. , Ng Pei‐Sze , Xu Xiaolin , Bergstrom Donald E. 
Published: June 2004

AbstractEndcaps may be either aromatic or aliphatic molecules that specifically cross‐link the 5′ end of one strand with the 3′ end of the complementary strand in a DNA duplex. Endcaps may be viewed as a replacement of the loop region nucleotides of a DNA hairpin, with the added advantage of increased thermal stability. An endcap is incorporated into the sequence during oligonucleotide synthesis. Three endcaps are described in this unit. The naphthalene diimide endcap prefers to base stack with GC base pairs. The terthiophene endcap has higher lipophilicity than the naphthalene diimide endcap and provides higher stability when stacked over an AT base pair. The 2,2′‐oxydiacetamide endcap provides lower enhancement in stability but a more rigid and well‐defined structure than the oligo(ethylene glycol) endcaps. Synthesis of endcapped oligonucleotides can be carried out using standard automated synthesis protocols with only minor modifications.
Engineering Terminal Disulfide Bonds Into DNA
Authors: Glick Gary D. 
Published: June 2004

AbstractThis protocol presents a simple and general means of modifying nucleic acids with disulfide cross‐links. These cross‐links serve as powerful tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross‐links have been used to study proteins. The chemistry described has been used to synthesize disulfide‐cross‐linked hairpins and duplexes, higher‐order structures such as triplexes, non‐ground‐state conformations, and tRNAs. since the cross‐links form quantitatively by mild air oxidation and do not purturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids.
Probing RNA Structure with Chemical Reagents and Enzymes
Authors: Ziehler William A. , Engelke David R. 
Published: June 2004

AbstractThis unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.
Probing DNA Structure with Hydroxyl Radicals
Authors: Tullius Thomas D. 
Published: June 2004

AbstractThe hydroxyl radical is a useful probe for studying the shape of the surface of a DNA molecule. Using this technique, fine details of DNA structure can potentially be revealed. This unit describes how to use the hydroxyl radical to generate a random cleavage pattern at the surface of the molecule, separate the broken DNA strands by polyacrylamide gel electrophoresis, and analyze the cleavage pattern to give an image of the surface of the molecule.
Optical Methods
Authors: Plum G. Eric 
Published: June 2004

AbstractEquilibrium optical melting curves are used in this unit to determine thermodynamic parameters of nucleic acid complex formation. This section contains all of the relevant equations and a discussion of which are most appropriate to a given situation. Additionally, procedures are given for making preliminary determinations of molar extinction coefficients and for determining the number of oligonucleotides in a complex. The section on extinction coefficients is particularly essential to anyone needing to know solution concentrations.
Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A. 
Published: June 2004

AbstractThis unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all‐atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics.
Molecular Modeling of Nucleic Acid Structure: Setup and Analysis
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A. 
Published: June 2004

AbstractThe last in a set of units by these authors, this unit addresses some important remaining questions about molecular modeling of nucleic acids. It describes how to choose an appropriate molecular mechanics force field; how to set up and equilibrate the system for accurate simulation of a nucleic acid in an explicit solvent by molecular dynamics or Monte Carlo simulation; and how to analyze molecular dynamics trajectories.
Determination of Binding Mode: Intercalation
Authors: Dedon Peter C. 
Published: June 2004

AbstractA small molecule can be assumed to bind to DNA by intercalation between base pairs if it causes lengthening and unwinding of the DNA helix and undergoes changes in its spectral properties, such as DNA‐induced hypochromism and quenching of its UV absorbance. DNA lengthening and unwinding can be determined from the change in viscosity of a solution of linear or plasmid DNA, respectively. Intercalation of a ligand can also be seen as a reduction in the UV/visible absorbance of the intercalator, as well as a shift in the absorbance maximum.
Chemistry of Minor Groove Binder–Oligonucleotide Conjugates
Authors: Kutyavin Igor , Lokhov Sergey , Lukhtanov Eugene , Reed Michael W. 
Published: June 2004

AbstractVarious types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB‐ODNs) with DNA are reviewed here. MB‐ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence‐specific DNA probes. Short MB‐ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB‐ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB‐ODNs allows for more stringent hybridization conditions to be used in DNA probe‐based assays. MB‐ODNs are especially useful in quantitative “real‐time” PCR assays since they bind efficiently during the high‐temperature primer extension cycle. The synthesis and biophysical chemistry of MB‐ODN conjugates are reviewed here. Four published structural classes of MB‐ODNs and their various dsDNA binding modes are discussed, and the well‐characterized DPI3‐type MB‐ODNs and their interactions with ssDNA target strands are described in detail.
Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries
Authors: Vant‐Hull Barry , Gold Larry , Zichi Dominic A. 
Published: June 2004

AbstractA new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal‐to‐noise ratios.
Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
Authors: Pollard Jack , Bell Sabine D. , Ellington Andrew D. 
Published: June 2004

AbstractPreparation of a random‐sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single‐stranded pool and conversion to a double‐stranded pool are presented as step‐by‐step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.
In Vitro Selection of RNA Aptamers to a Small Molecule Target
Authors: Jhaveri Sulay , Ellington Andrew 
Published: June 2004

AbstractThis unit describes the selection of aptamers that bind to a small molecule target from a single‐stranded RNA pool. Aptamers generated from this type of selection experiment can potentially function as receptors for small molecules for applications ranging from medical diagnostics to environmental monitoring. This unit describes two modes of selection, one by column filtration and one by batch selection.
Analysis of Oxidized DNA Fragments by Gel Electrophoresis
Authors: Awada Mohamad , Dedon Peter C. 
Published: June 2004

AbstractPolyacrylamide gel electrophoresis is used to define and quantify products of deoxyribose oxidation in DNA, based on the unique electrophoretic mobility of DNA fragments possessing deoxyribose oxidation products on their termini. This approach allows initial estimation of the chemistry. Once the chemical identity of damage products has been confirmed, this technique allows sensitive quantitation of the various damage products.
Probing RNA Folding Pathways by RNA Fingerprinting
Authors: Woodson Sarah A. 
Published: June 2004

AbstractThis unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution‐based methods. Conformers that have been separated and immobilized in the gel matrix can be used to studying catalytic activity with or without being eluted from the gel. The method can be applied to a wide varied of catalytic RNAs and RNA‐protein complexes.
Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea
Authors: Sosnick Tobin R. 
Published: June 2004

AbstractCD spectroscopy can be used to monitor RNA tertiary folding transitions that may not be observable by absorbance spectroscopy. With the use of computer‐controlled titrators, data can be acquired rapidly, and accurate thermodynamic properties can be obtained over a wide variety of conditions. Thus, CD spectroscopy provides a useful complement to site‐resolved or chemical modification methods.
Time‐Resolved Hydroxyl Radical Footprinting of RNA with X‐Rays
Authors: Woodson Sarah A. , Deras Michael L. , Brenowitz Michael 
Published: June 2004

AbstractRNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X‐ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.
Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways
Authors: Bevilacqua Philip C. , Turner Douglas H. 
Published: June 2004

AbstractThis overview 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.
Probing RNA Structural Dynamics and Function by Fluorescence Resonance Energy Transfer (FRET)
Authors: Walter Nils G. 
Published: June 2004

AbstractBiological function of RNA is often mediated by cyclic switching between several (meta‐)stable arrangements of tertiary structure. Fluorophore labeling of RNA offers a unique view into these folding and conformational switching events, since a fluorescence signal is sensitive to its molecular environment and can be continuously monitored in real time to produce kinetic rate information. This unit focuses on the practical implications of using fluorescence resonance energy transfer (FRET) to probe RNA structural dynamics and function. FRET is a particularly powerful fluorescence technique since, in addition to kinetic data, it provides insights into the structural basis of a conformational rearrangement. Protocols describe how to postsynthetically label RNA for FRET and how to acquire and analyze FRET data. Support protocols describe methods for deprotecting synthetic RNA and for purifying RNA by gel electrophoresis and HPLC. Considerations for selecting appropriate RNA, fluorophores, and labeling strategies are discussed in detail in the commentary.
Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates
Authors: Johnson David C. , Widlanski Theodore S. 
Published: June 2004

AbstractThis overview summarizes methodology used for the synthesis of nucleoside mono‐, di‐, and triphosphates. Selected techniques such as the Mitsunobu reaction, displacement reactions involving nucleoside 5′‐tosylates, “anion‐exchange” techniques, and phosphoramidite and phosphoramidate methodologies are highlighted. The chemistry of phosphorylation is detailed with respect to advantages and limitations under various conditions. Applicability of the methods toward the synthesis of analogs such as imidophosphates, phosphorothioates, and radiolabeled nucleotides is also addressed.
Characteristics of Nucleic Acids
Authors: Bergstrom Donald E. 
Published: June 2004

AbstractThis appendix is a compilation of information from Current Protocols in Molecular Biology (John Wiley & Sons, Inc.) and provides information about the major nucleoside/nucleotide constituents and structures for A‐, B‐, and Z‐DNA. Tables and figures provide (1) experimentally useful properties of the nucleoside/nucleotide building blocks; (2) chemical structures of the nucleosides; (3) aspects of nucleotide stereochemistry that are important to an understanding of base pairing and secondary structure; (4) alternative (non‐Watson‐Crick) base pairings; and (5) the three best characterized helix structures for DNA: A‐, B‐, and Z‐DNA.
Thin‐Layer Chromatography
Authors: Meyers C.L.F. 
Published: June 2004

AbstractTLC is used extensively in nucleic acid chemistry to monitor the progress of chemical reactions, to assay fractions collected from a larger chromatographic separation (e.g., column chromatography), and to determine optimal conditions prior to column chromatography. This unit describes methods for spotting test compounds onto a TLC plate, developing the plate in a suitable solvent system, visualizing the results, and calculating the retention factor (Rf). Candidate compounds can be co‐spotted for identification without relying on Rf values.
Extracranial Carotid Artery Disease
Authors: Haacke E. Mark , Kido Daniel , Tong Karen 
Published: June 2004

AbstractMagnetic Resonance Angiography (MRA) is a noninvasive means for studying the integrity of the vascular system. This unit presents a basic protocol describing an MRA technique for imaging the carotid arteries, time‐of‐flight MRA, and an for contrast‐enhanced MRA. A second basic protocol based on high‐resolution spin‐echo scanning is also described. This protocol can be used to study the vessel wall in more detail. The parameters given here are derived from experience at 1.5 T and may need to be altered slightly depending on the field strength and the equipment manufacturer. In particular, optimal TE may vary with different field strength.
Metastatic Intra‐Axial Neoplasia
Authors: Nusbaum Annette O. , Atlas Scott W. 
Published: June 2004

AbstractThe role of MRI imaging in the metastatic work‐up is to detect the spread of tumor to the brain parenchyma, and define the location. Intravenous contrast (gadolinium‐DTPA) provides the greatest sensitivity for detecting brain lesions and is almost always indicated except when there is no intravenous access. This unit presents a for imaging intra‐axial brain metastases and specific modifications are discussed for the case of metastatic brain tumors demonstrating evidence of hemorrhage. The sequences described in this unit are based on 1.5 T scanner (Echospeed GE Medical Systems, Milwaukee, Wisconsin), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Metastatic Extra‐Axial Neoplasia
Authors: Nusbaum Annette O. , Atlas Scott W. 
Published: June 2004

AbstractThere are a multitude of tumors that may spread to the extra‐axial compartment (i.e., meninges, skull base, and calvarium). The role of imaging in the search for metastases is to detect the full extent of and to correctly localize the disease. This unit presents basic protocols for imaging extra‐axial metastatic tumors. Specific modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (Echospeed GE Medical Systems, Milwaukee, Wisconsin), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Extra‐Axial Tumors
Authors: Nusbaum Annette O. , Atlas Scott W. 
Published: June 2004

AbstractExtra‐axial tumors most commonly arise from the meninges, calvarium, or skull base. Localization of a lesion as extra‐axial or extra‐cerebral in origin has significant clinical importance in terms of treatment planning and predicting prognosis. High‐resolution fat‐suppressed imaging is crucial for specific evaluation of the skull base, and correlation with computed tomography (CT) is often helpful in these cases. This unit presents basic MRI protocols for imaging extra‐axial tumors in specific locations; sequence modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (GE Medical Systems), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Meningitis
Authors: Auber Andrew E. , Belden Clifford 
Published: June 2004

AbstractMagnetic Resonance Imaging (MRI) in meningitis, as with most other forms of intracranial inflammatory or infectious diseases, is a powerful though largely non‐specific diagnostic tool. The technique is used to detect the presence of disease, and also complications related to the primary process, as well as assess for alternative diagnoses. For imaging these patients, a standard imaging protocol is utilized which includes gadolinium‐enhanced sequences. This unit presents a protocol based on diffusion MRI (dMRI), which can be employed if specific clinical situations require further clarification. The parameters given in this unit are derived from experience at 1.5T and may need to be altered slightly depending on the field strength available and the specific equipment manufacturer.
Cranial Nerves IX To XII
Authors: Evers Robert W. , Yousem David M. 
Published: June 2004

AbstractCranial nerves IX to XII are rarely affected by pathology compared with cranial nerves III, V, VII, and VIII. Nonetheless, their evaluation is challenging, since lesions of these nerves span the gamut from intracranial to extracranial sites. Imaging of these cranial nerves requires a focused approach based on clinical symptomatology and signs. This unit presents the basic protocol for imaging cranial nerves IX to XII. An alternate protocol is presented for cases where non‐neoplastic lesions are considered.