bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Brief History, Status, and Perspective of Modified Oligonucleotides for Chemotherapeutic Applications
Authors: Cook P. Dan 
Published: June 2004

AbstractThe advent of rapid and efficient methods of oligonucleotide synthesis has allowed the design of modified oligonucleotides that are complementary to specific nucleotide sequences in mRNA targets. Such modified oligonucleotides can be used to disrupt the flow of genetic information from transcribed mRNAs to proteins. This antisense strategy has been used to develop therapeutic oligonucleotides against cancer and various infectious diseases in humans. This overview reports recent advances in the application of oligonucleotides as drug candidates, describes the relationship between oligonucleotide modifications and their therapeutic profiles, and provides general guidelines for enhancing oligonucleotide drug properties.
Modification of the 5′ Terminus of Oligonucleotides for Attachment of Reporter and Conjugate Groups
Authors: Thuong Nguyen T. , Asseline Ulysse 
Published: June 2004

AbstractReporter and conjugate groups can be added directly to the 5′ terminus of oligonucleotides by appropriate modification. Conjugate groups can be used to increase the affinity of complementary strands, induce irreversible modification of target sequences, or enable sequences to recognize and permeate target cell membranes. This overview discusses the 5′ modifications that can be used and strategies for the covalent attachment of ligands to the modified oligonucleotides. Step‐by‐step protocols for attachment of conjugate groups are given elsewhere in the series.
Synthesis and Characterization of Chimeric 2‐5A‐DNA Oligonucleotides
Authors: Player Mark R. , Torrence Paul F. 
Published: June 2004

AbstractThis unit provides protocols for the synthesis and characterization of 2‐5A‐antisense nucleic acids. These chimeric oligonucleotides consist of 2′,5′‐phosphodiester‐linked oligoadenylates ligated to 3′,5′‐deoxyribonucleotides and are readily prepared using phosphoramidite chemistry on CPG solid supports. The 3′,5′‐deoxyribonucleotide functions as the antisense domain to target a given mRNA sequence, while the 2′,5′‐phosphodiester‐linked oligoadenylate serves to locally activate 2‐5A‐dependent RNase L, causing the targeted sequence to be cleaved.
Synthesis and Purification of Peptide Nucleic Acids
Authors: Braasch Dwaine A. , Nulf Christopher J. , Corey David R. 
Published: June 2004

AbstractPeptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.
Probing RNA Structure by Lead Cleavage
Authors: Pan Tao 
Published: June 2004

AbstractLead cleavage causes a transesterification reaction that breaks the 5′,3′‐phosphodiester backbone of RNA, leaving a 2′,3′‐cyclic phosphate and a 5′‐hydroxyl. Since the efficiency of the reaction at the 2′‐hydroxyl is related to steric and chemical constraints on particular 2′‐hydroxyls embedded in the RNA, this reaction can be used to examine the structure of individual nucleotides within an RNA molecule. It is a sensitive probe of tertiary RNA structure, provided that Pb2+‐binding sites are created in the tertiary structure.
Probing RNA Structures with Hydroxyl Radicals
Authors: Celander Daniel W. 
Published: June 2004

AbstractFe(II)‐EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar‐phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure. Because some positions become protected by tertiary folding, this reagent is useful for monitoring the global folding of RNA at equilibrium.
Chemical Reagents for Investigating the Major Groove of DNA
Authors: Rokita Steven E. 
Published: June 2004

AbstractChemical modification provides an inexpensive and rapid method for characterizing the structure of DNA and its association with drugs and proteins. Numerous conformation‐specific probes are available, but most investigations rely on only the most common and readily available of these. The major groove of DNA is typically characterized by reaction with dimethyl sulfate, diethyl pyrocarbonate, potassium permanganate, osmium tetroxide, and, quite recently, bromide with monoperoxysulfate. This commentary discusses the specificity of these reagents and their applications in protection, interference, and missing contact experiments.
Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting
Authors: Harris Dinari A. , Walter Nils G. 
Published: June 2004

AbstractThe function of an RNA molecule is determined by its overall secondary and tertiary structure. The tertiary structure is facilitated and stabilized by the interaction with metal ions. The current chapter offers a detailed protocol on the use of the lanthanide metal ion terbium(III) as a powerful probe of RNA structure and metal‐binding properties. When incubating RNA with low (micromolar) concentrations of terbium(III), specific backbone scission by partially deprotonated aqueous terbium(III) complexes can be used to detect high‐affinity metal‐binding sites, while incubation with high (millimolar) terbium(III) concentrations cleaves the RNA backbone preferentially at structurally accessible regions, providing a footprint of the RNA secondary and tertiary structure.
Calorimetry of Nucleic Acids
Authors: Pilch Daniel S. 
Published: June 2004

AbstractThis unit describes the application of differential scanning and isothermal titration calorimetry (DSC and ITC) to the study of the thermodynamics of nucleic acid structure. DSC is used to study order‐disorder transitions. A single DSC profile provides a wealth of thermodynamic and extrathermodynamic information: transition enthalpy, entropy, free energy, heat capacity, the state of the transition (two‐state vs. multistate), and the size of the cooperative unit. ITC is used to study hybridization of nucleic acids at constant temperature. Results can be used to determine the stoichiometry of the association reaction, the enthalpy of association, the equilibrium association constant, and the free energy and entropy of association. A thorough discussion is presented of the details required to obtain meaningful results, as well as relevant methods for analyzing the data produced.
Characterization of DNA Structures by Circular Dichroism
Authors: Bishop G. Reid , Chaires Jonathan B. 
Published: June 2004

AbstractCircular dichroism (CD) of nucleic acids is commonly used to provide a signature for a given secondary structure. CD is particularly powerful for monitoring structural changes resulting from changes in environmental conditions such as temperature, ionic strength, and pH. A number of commercially available instruments are available for the measurement of CD spectra. Since the details of instrumental operation differ for each machine, this unit provides a descriptive guide, rather than a strict protocol. It describes considerations that the experimentalist will face when attempting to examine nucleic acid structures by CD spectropolarimetry. Specific details are also given for cell filling and cleaning, and for instrumental calibration. Finally, several examples of CD spectra are given to provide useful reference points for a variety of nucleic acid structures, ranging from single‐stranded to triplex and quadruplex forms.
In Vitro Selection Using Modified or Unnatural Nucleotides
Authors: Knudsen Scott M. , Robertson Michael P. , Ellington Andrew D. 
Published: June 2004

AbstractThe use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.
Polyacrylamide Gel Electrophoresis (PAGE) of Synthetic Nucleic Acids
Authors: Andrus Alex , Kuimelis Robert G. 
Published: June 2004

AbstractProtocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.
Analysis and Purification of Synthetic Nucleic Acids Using HPLC
Authors: Andrus Alex , Kuimelis Robert G. 
Published: June 2004

AbstractHPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed‐phase HPLC allows a high‐capacity method for purification, and uses volatile buffer systems that simplify product recovery. Anion‐exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed‐phase and anion‐exchange HPLC, it can be used for oligonucleotides of up to ˜50 nt in length.
Cartridge Methods for Oligonucleotide Purification
Authors: Andrus Alex , Kuimelis Robert G. 
Published: June 2004

AbstractProtocols are given for purification of oligonucleotides by dimethoxytrityl‐sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean‐up by selective adsorption, and elution on solid‐phase media. Many of these products are prepackaged, single‐use cartridges or columns filled with affinity or size‐exclusion media.
Denaturing Polyacrylamide Gel Electrophoresis
Authors: Albright Lisa M. , Slatko Barton E. 
Published: June 2004

AbstractThin polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (<500 nucleotides) single‐stranded fragments of DNA or RNA that differ in length by as little as one nucleotide. Such gels are uniquely suited for nucleic acid sequence analysis, which is required, for instance, for all footprinting protocols. Thicker gels are often used to purify oligonucleotides. This appendix describes the pouring, running, and processing of a typical sequencing gel, which is 40 cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide.
Imaging Dural Sinus Thrombosis
Authors: Midyett F. Allan , Mukherji Suresh , Fisher Laurie 
Published: June 2004

AbstractThe acute onset of irreversible neurological deficit is referred to as a “stroke” and is the third leading cause of death and the major cause of adult long‐term disability in the United States. Obstruction of the venous sinuses by thrombosis accounts for ˜1% of strokes. Thrombosis of the superior saggital sinus is most frequent, with involvement of transverse, sigmoid, and cavernous sinuses occurring less often. Venous thrombosis may involve the dural sinuses, the deep venous system, and the superficial cortical veins separately or in any combination. This unit describes a for imaging dural sinuses by time‐of‐flight magnetic resonance venography (MRV).
Pituitary
Authors: Hallam Danial K. 
Published: June 2004

AbstractBenefiting from superior tissue contrast, multi‐planar capability and lack of bone artifact, MRI readily depicts complex anatomy in and about the pituitary gland. This unit presents three basic protocols for common indications relating to pathology of the sella and parasellar region. The protocols differ in emphasis more than in concept, and share a basic theme of thin slice high‐resolution imaging including the use of gadolinium. With the possible exception of dynamic imaging, all protocols may be readily performed on any MR scanner.
Paradigms for Clinical fMRI
Authors: Thulborn Keith R. , Davis Denise 
Published: June 2004

AbstractThis unit presents description of four basic clinical paradigms for clinical fMRI of language cmprehension, eye movement (visually guidede saccades), motor cortex (finger‐thumb apposition), and visual cortex. Given the neurosurgical concern with preservation of eloquent cortex, brain functions of particular interest are primary sensory (e.g., visual, auditory) and motor functions, and high level processing of language comprehension and expression. Compromise of these functions is usually readily apparent clinically and the quality of life of the patient is severely diminished. Functional MRI offers a means to locate these functions, thereby allowing the surgeon to plan on preserving these functions, or prepare the patient appropriately.
Cranial Nerve I
Authors: Evers Robert W. , Yousem David M. 
Published: June 2004

AbstractThis unit presents the basic protocol for imaging cranial nerve I. The olfactory bulbs and tracts mediate the sense of smell from the nasal cavity to the brain. Unfortunately they are located in a precarious position for MR imaging, above the air‐filled nasal cavity and ethmoid sinuses at a bone‐air‐soft tissue interface. This creates problems with susceptibility artifact. This issue, plus the very small size of the structures to be studied and the superimposed eye motion artifact makes imaging of the olfactory system a technical challenge.
Cranial Nerves III to VI
Authors: Evers Robert W. , Yousem David M. 
Published: June 2004

AbstractCranial nerves III, IV, V, and VI are small structures that travel in a reproducible manner from the midbrain and pons to the cavernous sinus and then to the orbit. While there are branches that course through other foramina of the skull, the emphasis in MRI is to evaluate the brainstem, the cavernous sinus, and the pericavernous regions for pathology. This unit present a basic protocol for imaging cranial nerves III to VI. Because the nerves run from a posterior to an anterior position, coronal scanning is ideal for visualizing the nerves in cross‐section. Thin sections and contrast enhancement are required to best visualize the diseases that affect these nerves. An alternate protocol is also discussed for the case when demyelinating etiologies for the cranial nerve deficits are considered.
Spinal Primary Neoplasia/Metastasis
Authors: Jinkins J. Randy , Stark David D. 
Published: June 2004

AbstractThis unit presents a basic protocol for conventional and fast spin echo imaging of spine for visualization of the spinal compartment to assess the extent and degree of spinal cord or cauda equina compression frequently associated with primary and metastatic neoplasia. An alternate protocol is presented for the cases e.g., neurofibromatosis with multiple bilateral neoplasms extending through the neural foramina, where a coronal acquisition may be helpful to analyze the perispinal tissues for tumor extension either inward to, or outward from, the central spinal canal.
Myocardial Perfusion and Viability
Authors: Jerosch‐Herold Michael , Stillman Arthur E. 
Published: June 2004

AbstractBoth first‐pass perfusion studies and imaging of delayed hyperenhancement can be combined in one patient exam. The MRI protocols for myocardial perfusion and viability assessment are presented together in this unit due to the complimentary information that is obtained with both protocols. Also, the imaging techniques used for both protocols are closely related. The first protocol assesses the functional severity of coronary artery lesions, while the second, used in addition to first basic protocol, is used in patient presents with symptoms of myocardial infarction or post‐coronary revascularization. The procedures are useful in determining the presence and extent of nonviable myocardium.
Nulling Signal of a Slice, Slice Select Profile, and Radiofrequency Power
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractThis unit discusses nulling of remaining signal from a RF pulse prior to the next RF pulse. This is to avoid mixing leftover signal with the signal generated from the next RF pulse. The slice select profile is also discussed in detail. The power of the RF pulse and its limit imposed by the Food and Drug Administration (FDA) is also discussed.Normal.
Short‐TR, Spoiled, Gradient Echo Imaging
Authors: Norman Cheng Yu‐Chung , Haacke E. Mark 
Published: June 2004

AbstractWhen a spin system is repeatedly disturbed by a fast repetition of RF pulses, the transverse magnetization after each new RF pulse approaches a steady‐state value which is smaller than the thermal equilibrium value. The spin system takes a finite number of pulses before this steady‐state is reached in a time that depends on both the T1 of the tissue and the flip angle of the RF pulse. Sequences utilizing a steady‐state approach can be broadly classified as steady‐state incoherent (SSI) and steady‐state coherent (SSC) sequences. The main difference between the two lies in whether the transverse magnetization is forced to zero or allowed to go naturally to steady‐state between successive RF pulses. As the nomenclature suggests, the SSI sequences are based on the elimination, or the “spoiling,” of any remnant transverse magnetization prior to the occurrence of each new RF pulse. This unit discusses the SSI sequences in detail.
Signal and Noise
Authors: Celik Azim , Lin Weili 
Published: June 2004

AbstractSignal and Noise (Azim Celik and Weili Lin, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). This unit discusses the basic signal detection technique in magnetic resonance imaging based on quadrature (i.e., measured in two orthogonal channels) technique. The detected signal is demodulated with respect to the Larmor frequency and digitized. The measured MR signal also includes noise. The noise in MR generally derives from the random fluctuations in the receive coil electronics and from the sample. Even though there are other sources of signal fluctuations such as digitization noise and pseudo‐random ghosting due to moving spins, these sources are minimized in an ideal experiment. The effect of these noises in MRI image reconstruction is explained in this unit.
Signal‐to‐Noise Ratio as a Function of Imaging Parameters
Authors: Celik Azim , Lin Weili 
Published: June 2004

AbstractSignal‐to‐Noise Ratio as a Function of Imaging Parameters (Azim Celik, General Electric Company, Milwaukee, Wisconsin and Weili Lin, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). The degree to which noise affects a measurement is generally characterized by the signal‐to‐noise ratio (SNR, as measured by the ratio of the voxel signal to the noise standard deviation). This unit describes the importance of SNR in describing image quality. SNR is the key parameter for determining the quality of any given imaging experiment. If the SNR is not high enough, it becomes impossible to differentiate tissues from one another or the background. The dependence of SNR on imaging parameters such as the number of repetitions, the number of k‐space samples (Nx, Ny, and Nz), the readout bandwidth, and voxel dimensions (Dx, Dy, and Dz) is explained in detail.
Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
Authors: Zeller Rolf 
Published: June 2004

AbstractThis unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while the alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.
Minipreps of Plasmid DNA
Authors: Engebrecht JoAnne , Brent Roger , Kaderbhai Mustak A. 
Published: June 2004

AbstractAlthough there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5‐ml tubes or 96‐well microtiter dishes, the boiling method, and a lithium‐based procedure. A support protocol provides information on storing plasmid DNA.
Preparing and Using M13‐Derived Vectors
Authors: Greenstein David , Besmond Claude 
Published: June 2004

AbstractMany vectors in current use are derived from filamentous phages. These vectors are used because DNA inserted into them can be recovered in two forms: double‐stranded circles and single‐stranded circles. This overview unit describes the lifecycle of filamentous phages along with the development and use of filamentous phage vectors.
Preparation of Genomic DNA from Plant Tissue
Authors: Richards Eric , Reichardt Mark , Rogers Sharon 
Published: June 2004

AbstractThis unit describes two methods for preparing genomic DNA from plant tissue. In the first method, plant cells are lysed with ionic detergent, treated with protease, and subsequently purified by cesium chloride (CsCl) density gradient centrifugation. The second method is based upon a series of treatments with the nonionic detergent cetyltrimethylammonium bromide (CTAB) to lyse cells and purify nucleic acid. Nucleic acid is recovered from the final CTAB solution by isopropanol or ethanol precipitation. The first method, although somewhat more lengthy, results in highly purified nucleic acid. The second method requires fewer manipulations, results in very high yields (˜10‐fold higher per gram fresh tissue depending on species and condition of starting material), and produces DNA that is less pure but nonetheless suitable in quality for use in many molecular biology manipulations.
DNA‐Dependent RNA Polymerases
Authors: Tabor Stanley 
Published: June 2004

AbstractThere are two types of DNA‐dependent RNA polymerases commonly used for nucleic acid research: E. coli RNA polymerase and the bacteriophage T7, T3, and SP6 RNA polymerases. This unit describes the uses and limitations of each class of RNA polymerase along with specific reaction conditions and applications.
Constructing Recombinant DNA Molecules by the Polymerase Chain Reaction
Authors: Elion Elaine A. 
Published: June 2004

AbstractThis unit describes the use of PCR to construct hybrid DNA molecules. The main objective is to give an overview of how PCR can be exploited to accomplish numerous cloning strategies; it is assumed that the reader is already familiar with basic molecular biology techniques including PCR amplification and subcloning. The outlines the PCR amplification and cloning strategies. A troubleshooting guide for problems most frequently encountered in PCR cloning, and three specific examples of this technique‐‐for creating (1) in‐frame fusion proteins, (2) recombinant DNA products, and (3) deletions and inversions by inverse PCR‐‐are also presented.