bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition
Authors: Leuck Michael , Wolter Andreas 
Published: June 2004

AbstractThe conjugation of maleimide reporter groups to 5′‐diene‐modified oligonucleotides via aqueous Diels‐Alder cycloaddition is described. Detailed basic protocols are provided for the preparation of a diene‐amidite (5′‐diene modifier), for the attachment of a diene group to the 5′‐terminus of oligonucleotides, and for the conjugation of maleimide reporter groups to diene‐modified oligonucleotides.
5′‐Iodination of Solid‐Phase‐Linked Oligodeoxyribonucleotides
Authors: Kool Eric T. , Miller Gregory P. 
Published: June 2004

Abstract5′‐Iodinated oligodeoxyribonucleotides readily react with 3′‐phosphorothioated DNA in the presence of a complementary template to yield a conjugate that is identical to natural DNA in every respect except that one oxygen atom in the phosphodiester backbone is replaced by a sulfur atom. The 5′‐iodo group is easily converted to a variety of other functional groups and will quickly react with thiol‐containing labels to yield stable thioether conjugates. This unit presents manual and automated procedures for converting the 5′‐hydroxyl of protected CPG–bound oligodeoxyribonucleotides to an iodo group and for releasing and purifying the products.
Uridine 2′‐Carbamates: Facile Tools for Oligonucleotide 2′‐Functionalization
Authors: Korshun Vladimir A. , Stetsenko Dmitry A. , Gait Michael J. 
Published: June 2004

AbstractA facile method for preparation of uridine 2′‐carbamate derivatives based on reaction of 3′,5′‐disilyl‐protected uridine with 1,1′‐carbonyldiimidazole followed by treatment with an aliphatic amine is presented. A phosphoramidite monomer suitable for automated oligonucleotide synthesis is obtained in a few steps. The compounds are useful for the introduction of various labels and modifications into an oligonucleotide chain. Although 2′‐carbamate modification is somewhat destabilizing for DNA‐DNA and DNA‐RNA duplexes, it is suitable for the direction of ligands into the minor groove of duplexes or at non‐base‐paired sites (e.g., loops and bulges) of oligonucleotides. Pyrene‐modified oligonucleotide 2′‐carbamates show a considerable increase in fluorescence intensity upon hybridization to a complementary RNA (but not DNA).
Engineering Specific Cross‐Links in Nucleic Acids Using Glycol Linkers
Authors: O'Dea Timothy , McLaughlin Larry W. 
Published: June 2004

AbstractOne of the most convenient methods for generating oligonucleotides possessing intra‐ or interstrand cross‐links is through incorporation of oligoethylene glycol bridges by solid‐phase synthesis. The reagents are commercially available or can be synthesized in a few easy synthetic steps. Unlike many other DNA and RNA cross‐links, aspects of the structural and thermodynamic impact of modifying nucleic acids with oligoethylene glycols have been studied. This unit covers protection, phosphitylation, and preparation of the glycol linker for oligonucleotide synthesis.
Engineering Disulfide Cross‐Links in RNA Via Air Oxidation
Authors: Maglott Emily J. , Glick Gary D. 
Published: June 2004

AbstractThis unit presents protocols for the synthesis of alkylthiol‐modified ribonucleosides, their incorporation into synthetic RNA, and the formation of intramolecular disulfide bonds in RNA by air oxidation. The disulfide bonds can be formed in quantitative yields between thiols positioned in close proximity by virtue of either the secondary or tertiary structure of the RNA. Disulfide cross‐links are useful tools to probe solution structures of RNA, to monitor dynamic motion, to stabilize folded RNAs, and to study the process of tertiary structure folding.
Use of Electrophilic Substitution to Form Site‐Specific Cross‐Links in DNA
Authors: Pires Richard M. , Coleman Robert S. 
Published: June 2004

AbstractThis unit describes the synthesis, purification, and analysis of modified oligodeoxynucleotides containing 4‐thio‐2′‐deoxyuridine, and the use of these modified oligodeoxynucleotides in conjunction with bis‐electrophilic tethers to form site‐specific cross‐links with a complementary DNA target. These modified oligodeoxynucleotides provide a flexible tool for studies directed at biophysical investigation of oligonucleotide structure, as well as investigation of DNA hybridization, stability, and sequence‐dependent base nucleophilicity.
Probing Nucleic Acid Structure with Shape‐Selective Rhodium and Ruthenium Complexes
Authors: Jackson Brian A. , Barton Jacqueline K. 
Published: June 2004

AbstractIn this unit, transition metal complexes are used as photochemical probes for the structure of RNA and DNA. The transition metal ion provides a rigid substitutionally inert framework and an octahedral geometry for ligand coordination. The complexes can be constructed to define shapes, symmetries, and functionalities that complement those of the nucleic acid target. Complex formation is easily detected by light‐induced nucleic acid cleavage. The modular construction of the complexes makes it possible to generate probes to examine a wide variety of structural characteristics of nucleic acids.
Probing Nucleic Acid Structure with Nickel‐ and Cobalt‐Based Reagents
Authors: Rokita Steven E. , Burrows Cynthia J. 
Published: June 2004

AbstractThe use of nickel and cobalt reagents is presented for characterizing the solvent exposure of guanine residues in DNA and RNA. These reagents promote guanine oxidation in the presence of a peracid such as monopersulfate, and the extent of reaction indicates the steric and electronic environment surrounding the N7 and aromatic face of this residue. Since oxidation does not itself perturb target structure or induce strand scission, it is coupled with fragmentation by treatment with piperidine (for smaller polynucleotides) or termination of primer extension (for larger polynucleotides).
Molecular Modeling of Nucleic Acid Structure
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A. 
Published: June 2004

AbstractThis unit is the first in a series of four units covering the analysis of nucleic acid structure by molecular modeling. This unit provides an overview of computer simulation of nucleic acids. Topics include the static structure model, computational graphics and energy models, generation of an initial model, and characterization of the overall three‐dimensional structure.
Methods to Crystallize RNA
Authors: Ferré‐D'Amaré Adrian R. , Doudna Jennifer A. 
Published: June 2004

AbstractPreparation of suitably large and well‐ordered single crystals is usually the rate‐limiting step in the determination of the three‐dimensional structure of RNAs and their complexes with proteins by X‐ray crystallography. This unit discusses a variety of experimental considerations for obtaining crystals of RNAs and RNA‐protein complexes. Topics include design of crystallizable constructs, screening, and optimization of crystallization conditions.
Recent Advances in RNA Structure Determination by NMR
Authors: Hennig M. , Williamson J.R. , Brodsky A.S. , Battiste J.L. 
Published: June 2004

AbstractDespite recent advances in the solution of NMR structures of RNA and RNA‐ligand complexes, the rate limiting step remains the gathering of a large number of NOE and torsion restraints. Additional sources of information for structure determination of larger RNA molecules have recently become available, and it is possible to supplement NOE and J‐coupling data with the measurement of dipolar couplings and cross‐correlated relaxation rates in high‐resolution NMR spectroscopy.
Molecular Modeling of Nucleic Acid Structure: Energy and Sampling
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A. 
Published: June 2004

AbstractAn overview of computer simulation techniques as applied to nucleic acid systems is presented. This unit expands an accompanying overview unit (UNIT here) by discussing methods used to treat the energy and sample representative configurations. Emphasis is placed on molecular mechanics and empirical force fields.
A Competition Dialysis Assay for the Study of Structure‐Selective Ligand Binding to Nucleic Acids
Authors: Chaires Jonathan B. 
Published: June 2004

AbstractUnique DNA structures represent potential targets for small molecules, and provide a promising new avenue for drug development. However, attempts to rationally design small molecules that bind selectively to a particular DNA structure have been hampered by the lack of a rapid and convenient assay for structural selectivity. Determination of structure‐selective ligand binding using competition dialysis is described in this unit. The competition dialysis assay is simple, straightforward, and rapid once stock solutions of the nucleic acid structures of interest have been prepared as described.
In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization
Authors: Jhaveri Sulay D. , Ellington Andrew D. 
Published: June 2004

AbstractThis unit describes the selection of aptamers from a pool of single‐stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially function as protein inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate a ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.
Analysis of Oligonucleotides by Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry
Authors: Chou Chau‐Wen , Limbach Patrick A. 
Published: June 2004

AbstractMALDI‐MS is one of the most useful techniques available for determining biomolecule mass. It offers high mass accuracy, good sensitivity, simplicity, and speed. Because singly charged ions of oligonucleotides are typically observed, MALDI‐MS spectra are easy to interpret. This unit presents protocols for sample preparation and purification, matrix preparation, and matrix/analyte sample preparation. It provides an introduction to the instrumentation and its calibration, and a discussion of some of the useful applications of MALDI‐MS analysis in the study of oligonucleotides. This technique is typically used for 120‐mer or smaller oligonucleotides.
Analysis of Oligonucleotides by Electrospray Ionization Mass Spectrometry
Authors: Polo Lenore M. , Limbach Patrick A. 
Published: June 2004

AbstractBecause of the high molecular weights and thermal lability of biomolecules such as nucleic acids and protein, they can be difficult to analyze by mass spectrometry. Such analyses require a “soft” ionization method that is capable of generating intact molecular ions. In addition, most mass analyzers have a limited upper mass range that is not sufficient for studying these large molecules. ESI‐MS can be used to analyze molecules with a molecular weight that is larger than the mass‐to‐charge ratio limit of the analyzer. This unit describes how ESI allows for analysis of high‐molecular‐weight compounds through the generation of multiply charged ions in the gas phase. It discusses analyzer configurations, solvent selection, and gives protocols for sample preparation. For applications of ESI‐MS, the unit discusses molecular weight determination and gives protocols for sequencing and for analyzing oligonucleotide modifications.
Overview of Purification and Analysis of Synthetic Nucleic Acids
Authors: Andrus Alex , Kuimelis Robert G. 
Published: June 2004

AbstractSynthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.
Base Composition Analysis of Nucleosides Using HPLC
Authors: Andrus Alex , Kuimelis Robert G. 
Published: June 2004

AbstractIn this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed‐phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.
Capillary Electrophoresis of DNA
Authors: Smith Alan , Nelson Robert J. 
Published: June 2004

AbstractCapillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single‐base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.
Rapid Magnesium Chelation as a Method to Study Real‐Time Tertiary Unfolding of RNA
Authors: Maglott Emily J. , Glick Gary D. 
Published: June 2004

AbstractThis unit describes a method to measure the unfolding of RNA tertiary structure on a millisecond time scale. A stopped‐flow spectrophotometer is used to measure the rate of unfolding induced by the addition of EDTA to an RNA whose tertiary structure has been stabilized in the presence of magnesium ions. Using this methodology, rate constants for unfolding of tertiary or secondary structure can be obtained over a range of temperatures, and these values can be used to construct Arrhenius and Eyring plots, from which activation energy, Arrhenius pre‐exponential factor, and enthalpy and entropy of activation can be obtained. These data provide information about the energy of the transition state and the energy barriers between secondary and tertiary structure, which is necessary for predicting RNA tertiary structure from secondary structure.
Nucleoside and Nucleotide Nomenclature
Authors: Secrist John A. 
Published: June 2004

AbstractCurrent nomenclature in the area of nucleosides, nucleotides, and nucleic acids comprises a mixture of (1) common names that have gained official recognition, (2) guidelines that have been derived and officially recommended by the International Union of Pure and Applied Chemistry (IUPAC)/International Union of Biochemistry and Molecular Biology (IUBMB), and (3) evolving usage that is derived by individual scientists and laboratories and subjected to peer review through publication. A working group was commissioned in 1998 by IUBMB to review guidelines for nucleotide (including oligonucleotide) nomenclature. As those guidelines are developed and made available, they will be referenced in future updates of this appendix. The main purpose of this appendix is to provide pertinent references that will direct the reader to the relevant guidelines or evolving nomenclature as described in the literature. When additional suggestions or guidance are appropriate, those comments are included as well.
Rule Out (R/O) Intracranial Aneurysm
Authors: Grant P. Ellen , Gonzalez R. Gilberto , Schaefer Pamela W. 
Published: June 2004

AbstractWhen imaging patients for intracranial aneurysm, the goals are: (1) to assess the contour of the intracranial arteries, particularly in he regions of the ACOM (anterior communicating artery), PCOM (posterior communicating artery), ICA (internal carotid artery) bifurcation, MCA (middle cerebral artery) trifurcation, basilar tip, and PICA (posterior inferior cerebellar artery); (2) to assess the anatomy of the Circle of Willis and direction of flow, and; (3) to determine if there is evidence of a recent subarachnoid bleed. This unit describes a that can be used for standard imaging of aneurysm in stable patients. An is described for situations when there is concern for vasospasm and infarction.
High‐Resolution Multi‐Contrast MRI of the Carotid Artery Wall for Evaluation of Atherosclerotic Plaques
Authors: Yarnykh Vasily L. , Yuan Chun 
Published: June 2004

AbstractCarotid atherosclerosis is an important medical condition associated with a high potential risk of stroke. In addition to the degree of artery stenosis, the size and tissue composition of the atherosclerotic plaque are thought to be important factors for disease management and prognosis. This unit presents a protocol for high‐resolution multi‐contrast MRI of the carotid artery wall. The protocol employs a combination of bright‐blood magnetic resonance angiography and black‐blood imaging with different contrast weightings (T1, T2, and proton‐density contrasts) in order to provide comprehensive characterization of the atherosclerotic lesion. The images obtained with the protocol presented here can be used to identify basic tissue components of the atherosclerotic plaque (fibrous matrix, lipid core, calcificates, and hemorrhage) and to conduct morphological measurements of plaque size and distribution.
Imaging Cavernous Malformations
Authors: Midyett F. Allan , Fisher Laurie , Mukherji Suresh 
Published: June 2004

AbstractThis unit describes a for evaluating cavernous malformations (CMs). CMs represent ˜10% to 15% of vascular malformations. They consist of enlarged sinusoidal vascular spaces that have thin walls devoid of smooth muscle and normal endothelium. These form compact masses within central nervous system (CNS) parenchyma or associated structures without normal interspersed tissue. The thin walls lack normal endothelium and are prone to leakage of blood elements.
Intra‐Axial Primary Brain Tumors
Authors: Nusbaum Annette O. , Atlas Scott W. 
Published: June 2004

AbstractThe majority of primary brain tumors in adults are found in the supratentorial compartment, while tumors in pediatric patients are usually infratentorial in location. This unit presents basic protocols for imaging all types of primary intra‐axial brain tumors, whether infiltrative (i.e., astrocytoma, oligodendroglioma, lymphoma) or circumscribed (i.e., ganglioglioma, cystic astrocytoma). Specific modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (Echospeed GE Medical Systems), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Monitoring Tumor Therapy
Authors: Nusbaum Annette O. , Atlas Scott W. 
Published: June 2004

AbstractThis unit presents the preferred protocol for imaging the brain following the institution of chemotherapy, radiation therapy, or surgery for brain tumors and specific modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (Echospeed GE Medical Systems), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Brain Abscess
Authors: Auber Andrew E. , Belden Clifford 
Published: June 2004

AbstractMagnetic Resonance Imaging (MRI) in cerebral abscess, as with most other forms of intracranial inflammatory or infectious diseases, is a powerful though largely nonspecific diagnostic tool. This unit presents a variant of a previously published standard imaging protocol, to include gadolinium‐enhanced sequences for imaging of these patients. Several optional sequences, including diffusion (dMRI), perfusion (pMRI), and spectroscopic (MRS) sequences are outlined that can be employed should patient tolerance allow and if specific clinical situations require further clarification. The parameters given in this unit are derived from experience at 1.5 T and may need to be altered slightly depending on the field strength available and the specific equipment manufacturer.
Traumatic Brain Injury
Authors: Auber Andrew E. , Belden Clifford 
Published: June 2004

AbstractMagnetic Resonance (MR) imaging of the brain following head injury is used in two distinct clinical contexts, (1) acutely, within days of the injury, to evaluate an unexplained neurologic deficit or to obtain prognostic information, and (2) chronically, to assess the degree of brain injury and explain neurologic or neuropsychologic findings. In this unit, two basic protocols are presented, one for acute imaging and the other for chronic imaging. Advanced MR imaging sequences, such as MR spectroscopy (MRS) and diffusion‐weighted (DW) imaging can provide additional prognostic information in the acute setting and are also described. MR angiography (MRA) and direct vessel wall imaging techniques are mentioned briefly.
Dementia
Authors: Hallam Danial K. , Salamon‐Muramaya Noriko 
Published: June 2004

AbstractThis unit presents specific protocols intended to maximize the contribution of MRI to the clinical evaluation of dementia. Each protocol has, as its foundation, a whole‐brain screen designed to evaluate for treatable causes of dementia. Reflecting the frequent overlap of vascular disease with dementing illness, the screening protocol also serves as an optimal evaluation of the patient with suspected vascular dementia. Additional sequences or modifications are then used to answer specific clinical questions. 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.
Quality Assurance for Clinical fMRI
Authors: Thulborn Keith R. , Davis Denise 
Published: June 2004

AbstractThe functional MRI (fMRI) procedure has several sources of variance that determine the success of the examination. These include the scanner, patient, and paradigm. As blood oxygenation level dependent (BOLD) contrast is a small effect, high signal‐to‐noise performance is mandatory. Because the preparation of a functional activation map requires averaging multiple images over time, the scanner must produce high temporal stability of the signal intensity. This unit presents the for achieving scanner stability. There are many determinants of such performance but not all possibilities need to be checked separately. An adequate approach has been to verify total system performance under the conditions of a functional MRI study on a phantom. This testing is done daily prior to patient studies.
Cranial Nerves VII To VIII
Authors: Evers Robert W. , Yousem David M. 
Published: June 2004

AbstractCranial nerves VII and VIII are easily visualized within cerebrospinal fluid (CSF) in most patients and are the nerves most commonly involved with neurogenic neoplasms. The reasons for performing contrast‐enhanced scans are explained in this unit, but revolve around pathologies other than schwannomas of the nerves.
Congenital Heart Disease
Authors: Woodard Pamela K. , Zheng Jie 
Published: June 2004

AbstractCardiac MRI plays a pivotal role in both anatomical and functional assessment of shunts, admixture lesions, transpositions, and the surgical correction of these lesions. This unit presents basic techniques for the evaluation of congenital heart disease. While sequence parameters described are meant to be as generic as possible, parameters are most appropriate for the Siemens 1.5 T Vision or Symphony, and may need to be altered for magnets of different field strengths and manufacturers.
MRI of the Liver
Authors: Fisher Laurie , Semelka Richard , Wilber Kathy 
Published: June 2004

AbstractMRI provides comprehensive information on the full range of liver diseases, including congenital abnormalities, benign and malignant focal liver lesions, and diffuse liver disease. This unit presents a protocol that incorporates various types of T1‐and T2‐weighted sequences, including transverse and coronal data acquisition, and the routine use of intravenous gadolinium.