bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of Second Messengers in Signal Transduction: cAMP and Inositol Phosphates
Authors: Gusovsky Fabian
Published: June 2004

cAMP acts as an intracellular mediator of hormone action and the importance of accurate quantitative determination of cAMP levels in cells and tissues is widely recognized. The most utilized procedures for the determination of adenylate cyclase activity in membranes are described here for measuring the conversion of [alpha‐32P]ATP into [32P]cAMP after a two‐step chromatographic separation. Also critical in signal tranduction is phosphoinositide turnover, which is linked to receptor activation resulting from changes in cytosolic calcium concentrations. Phosphoinositide turnover can be measured as described in this unit by labeling phospholipid pools with [3H]‐inositol and then analyzing for tritiated inositol phosphates.
Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic Microsensor
Authors: Malinski Tadeusz , Huk Igor
Published: June 2004

This unit describes the preparation and applications of porphyrinic sensors for quantitative measurement of nitric oxide (NO) in single cells and in tissues. The determination of NO is based on the electrochemical oxidation of NO on a carbon fiber electrode covered with a thin layer of a conducting polymeric metalloporphyrin catalyst, overlaid with another thin film of Nafion, a cation exchange material. The electric current generated during NO oxidation on the surface of the polymeric porphyrin is linearly proportional to the concentration of NO, so this current is used as an analytical signal which can be measured in either the amperometric or the voltammetric mode. Both methods provide a quantitative signal. This unit describes the electrochemical setup for measurement of NO in single cells and tissue. Support protocols describe porphyrin synthesis, sensor preparation, and sensor calibration.
Locomotor Behavior
Authors: Pierce R. Christopher , Kalivas Peter
Published: June 2004

Locomotor activity can be used to elucidate basic scientific questions, as the neural circuitry mediating the behavioral effects of different drug classes can be determined by direct pharmacological stimulation of specific brain nuclei. Locomotor activity is also used to assess the neural bases of ethological and biological rhythms. Methods for evaluating locomotor behavior in rodents fall into two general categories: direct observational techniques and automated monitoring of behavior. The first technique described in this unit consists of direct observation using interval and ordinal scales. The second approach is based on the measurement of locomotor activity with photocell‐based automated monitoring systems. Other methods used to quantify locomotor activity include video‐based systems, rotometers, and running wheels. The protocols in this unit are designed for rat subjects, although they can easily be adapted for use with mice and other rodents.
Social Learning of Food Preferences in Rodents: Rapid Appetitive Learning
Authors: Galef Bennett G.
Published: June 2004

A procedure is described for quantitating the transfer of information about inducing increased intake of distinctively‐flavored foods or fluids among common laboratory rodents. The method provides a simple, efficient, non‐invasive way to produce robust, long‐lasting changes in appetitive behaviors of laboratory rodents that can be used in studies of the neuroanatomical, neurochemical or genetic substrates of learning and memory.
Assessment of Learning and Memory Using the Autoshaping of Operant Responding in Mice
Authors: Barrett James E. , Vanover K.E.
Published: June 2004

This unit describes the use of an automated procedure for developing an operant response (“autoshaping”) in the mouse. The method has applications in the study of the acquisition of behavior (learning) as well as for the assessment of memory or retention of that task.
Sham Feeding in Rats with Chronic, Reversible Gastric Fistulas
Authors: Smith Gerard P.
Published: June 2004

Sham feeding occurs when ingested food is prevented from accumulating in the stomach and small intestine by surgical intervention. Sham feeding permits the investigation of the orosensory controls of meal size in the absence of postingestive controls. This unit details preoperative preparation, surgery, postoperative care and maintenance, and test procedures for the simplest and most widely used method, which involves implantation of a gastric cannula in a rat to provide a chronic, reversible gastric fistula.
Conditioned Flavor Aversions: Assessment of Drug‐Induced Suppression of Food Intake
Authors: Riley Anthony L.
Published: June 2004

Administration of a drug following ingestion of a novel food or solution often suppresses subsequent intake of the new food or solution. This suppression is associative, in that consumption is not suppressed when there is no temporal relationship between consumption and drug administration. The robust nature of aversion learning has made this procedure a sensitive and widely used behavioral index of drug side effects. The procedures described in this unit are suitable for work with rodents, and may require modifications, e.g., in presentation of the ingesta and drug for other species. Familiar and novel foods may be used instead of solutions, with similar results.
Measurement of Startle Response, Prepulse Inhibition, and Habituation
Authors: Geyer Mark A. , Swerdlow Neal R.
Published: June 2004

The startle response is comprised of a constellation of reflexes elicited by sudden, relatively intense stimuli. The startle reflex is useful for studying fundamental properties of nervous function ranging from neurophysiological and anatomical relationships within the pons and reticular formation to forebrain regulation of complex behavioral states and cognitive processes. This unit presents protocols for measurement of the startle response in rats to acoustic stimuli, along with modifications encompassing an experimental manipulation such as drug treatment and measures of habituation of startle; a method for measuring prepulse inhibition (PPI) of startle; and specialized rat handling and calming techniques that supplement the startle protocols.
Rodent Models of Depression: Learned Helplessness Using a Triadic Design in Rats
Authors: Durgam Robert C.
Published: June 2004

Certain types of human depression are precipitated by stressful life events, and vulnerable individuals experiencing these stressors may develop clinical depression. Understanding the neurobiology of stress vulnerability (depression) as well as stress resiliency (coping) is critical for guiding the development of novel pharmacotherapeutic agents for stress‐related disorders such as depression in humans. The use of a triadic design (escapable shock, yoked‐inescapable shock and restrained control) allows the investigator to examine the various sequella of stress exposure, while manipulating and quantifying the impact of psychological dynamics of stress such as active behavioral coping (i.e., stress control). Both escape and yoked subjects are exposed to the identical amount, intensity, pattern and duration of stress. The critical distinction between these two groups is that the escape group has the opportunity to terminate the shock stress by turning a wheel at the front of a chamber, while wheel‐turning for the yoked subject is of no consequence. Any difference observed between the escape and yoked subjects is a result of the effects of coping, rather than stress exposure per se. The restrained group is included to control for the effects of handling. Any differences between this group and the escape and yoked subjects reflects the impact of stress per se.
Rodent Models of Depression: Learned Helplessness Induced in Mice
Authors: Anisman Hymie , Merali Zul
Published: June 2004

Uncontrollable stressors induce a variety of behavioral disturbances that are in many ways reminiscent of the symptoms that characterize clinical depression. These deficits are evident across a range of species, including mice. Given the increasing focus on genetic techniques involving mice to identify the mechanisms subserving these behavioral disturbances (e.g., recombinant, knockout, and transgenic strains), it is of particular interest to provide a detailed description of the method to induce behavioral deficits in response to uncontrollable stressors. This unit describes the procedure used to assess the effects of controllable and uncontrollable shock on subsequent shock escape performance in mice using an escape‐delay procedure.
Fear‐Potentiated Startle in Mice
Authors: Falls William A.
Published: June 2004

Pavlovian fear conditioning is frequently used to assess the behavioral, physiological, genetic and molecular correlates of learning and memory. In the typical Pavlovian conditioned fear procedure a neutral stimulus, such as a tone, is paired with a mildly aversive stimulus such as a foot shock. The tone conditioned stimulus (CS) comes to elicit a variety of behaviors that are indicative of learned fear. One of the more prominent of these behaviors is a potentiated acoustic startle response. While fear‐potentiated startle in mice is qualitatively similar to that in rats, the stimulus parameters and procedures for producing optimum fear‐potentiated startle in mice differ considerably from those used in rats. Procedures outlined in this unit include initial assessment of startle, fear conditioning and fear‐potentiated startle testing. Special attention is paid to the parameters that affect the magnitude of fear‐potentiated startle and procedures designed to systematically examine these parameters are included.
Traumatic Brain Injury in the Rat Using the Fluid‐Percussion Model
Authors: Ling Geoffrey S.F. , Garcia‐Pinto Patricia
Published: June 2004

Traumatic brain injury is a leading cause of death and disability, particularly among young adults. During closed head trauma, the injury process is initiated by the impact of the brain against the inner table of the calvarium. Subsequently, there is prompt initiation of a complex biochemical, cellular, and physiological injury cascade that may take days to complete. From a functional standpoint, this culminates in neurologic dysfunction and, if severe, death. This unit describes an impact‐induced brain trauma model in rats which replicates nonpenetrating head injury. It does not model either penetrating or ischemic brain injuries.
Preclinical Models of Hepatic Encephalopathy
Authors: Basile Anthony S. , Mullen Kevin
Published: June 2004

Parkinson's disease is a neurodegenerative disease in which pigmented midbrain neurons progressively die producing a dopamine (DA) deficit in the striatum which manifests as an akinetic movement disorder. Experimentally induced striatal DA depletion in animals is a valid model of parkinsonism. The capacity of certain substances to damage catecholaminergic neurones has been used for a long time to produce DA deficiency in animals. This unit focuses on methods for inducing parkinsonism using the neurotoxins MPTP and 6‐hydroxy dopamine and methods for evaluating the animals. Other models are briefly reviewed.
Rodent Models of Global Cerebral Ischemia
Authors: O'Neill Michael J. , Clemens James A.
Published: June 2004

Brain damage after stroke and head injury remains a huge clinical problem. In stroke, the initial cause of the damage is a blockage in a blood vessel (often the middle cerebral artery) and this sets off several pathways that ultimately lead to cell death. Recent studies have demonstrated that several new mechanisms are involved in neuronal death and this has led to an increase in research into novel molecules that might prevent brain damage or improve recuperation post‐stroke. There are several models of global cerebral ischemia. Two of the most widely‐used models are discussed in detail in UNIT here, the gerbil bilateral carotid artery occlusion (BCAO) model and rat 4‐vessel occlusion (4‐VO) model. Additionally, several models of focal cerebral ischemia have been developed to mimic the effects of human stroke. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.
Overview of Rodent Models for Obesity Research
Authors: Tschöp Matthias , Heiman Mark L.
Published: June 2004

Animal obesity models differ widely in type and extent of obesity. They are either based on environmental factors (e.g., high‐fat diet‐induced obesity), spontaneous mutants (i.e., ob/ob mice), genetically engineered animals (e.g., mice with melanocortin receptor subtype‐4 gene disruption (knock‐out), or mechanical intervention (e.g., chemical lesion of the ventromedial hypothalamus). This unit reviews available rodent models to study obesity and attempts to highlight the greatest utility for each model.
Models of Neuropathic Pain in the Rat
Authors: Bennett Gary J. , Chung Jin Mo , Honore Marie , Seltzer Ze'ev
Published: June 2004

There are now three models of neuropathic pain in the rat that are in widespread use: the chronic constriction injury, the partial sciatic ligation model, and the spinal nerve ligation model. The procedures to create these models and the behavioral assays used to quantify the resulting abnormal pain sensations are described in this unit.
Place Preference Test in Rodents
Authors: Roux Sylvain , Froger Christelle , Porsolt Roger D. , Valverde Olga , Maldonado Rafael
Published: June 2004

Conditioned place preference is a behavioral model currently used to measure the rewarding properties induced by the administration of a drug. In this paradigm, the rewarding properties of a compound are associated with the particular characteristics of a given environment. Due to the recent availability of multiple lines of genetically modified mice, this unit includes procedures for testing place preference in both rats and mice.
Transfection by Electroporation
Authors: Potter Huntington
Published: June 2004

Electroporation‐‐the use of high‐voltage electric shocks to introduce DNA into cells‐‐can be used with most cell types,yields a high frequency of both stable transformation and transient gene expression,and,because it requires fewer steps,can be easier than alternate techniques. In this unit,the basic protocol describes the electroporation of mammalian cells and an alternate protocol outlines modifications for preparation and transfection of plant protoplasts.
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David
Published: June 2004

This unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated for reasons of convenience. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols provide modifications to the basic protocol that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low‐molecular‐weight oligonucleotides and triphosphates.
Preparation of an Iodinated Radioligand
Authors: True C. Diane
Published: June 2004

Radioactive tracers are useful for receptor measurement and for radioimmunoassays because of the ease with which radioactive ligands are detected. Typical receptor measurement methods include whole tissue receptor assays, autoradiography, and cell and membrane binding assays. The isotopes most commonly used in receptor measurement studies are tritium, carbon‐14, and iodine‐125, with the choice of isotope most often based on the specific activity required and the sensitivity and type of detection system available. Its high specific activity and easy detection make I‐125 ideal for labeling large peptides and proteins. While the short half‐life (60 days) of iodine requires frequent replacement with freshly iodinated compounds, this is also an advantage, as unused portions of radioligand can be held for decay and disposed of as nonradioactive waste. This unit describes the iodination of proteins or peptides using the 125I‐labeled Bolton Hunter reagent, a convenient, easy‐to‐use, nonoxidizing labeling reagent.
High Precision Stereotaxic Surgery in Mice
Authors: Athos Jaime , Storm Daniel R.
Published: June 2004

This unit provides protocols for cannulation and site‐specific central microinjection of mice using a recently developed high‐precision stereotaxic frame. The construction of cannulae, wire plugs and injection needles are also described.
Internet Resources for Genomic, Bioinformatics, and Medical Genetics Information
Authors: Phillips Tamara J. , Belknap J.K. , Hitzemann R.J. , Buck K.J. , Cunningham C.L. , Crabbe J.C.
Published: June 2004

Internet Resources for Genomic, Bioinformatics, and Medical Genetics Information (Tamara J. Phillips, Veterans Affairs Medical Center, Portland, Oregon). Many diseases are influenced by multiple genetic and environmental factors. Identifying the genes contributing to the probability of developing such diseases poses an extraordinary challenge because each gene may have a small influence. Further, in the presence of other influential genes and environmental factors, the impact of a single gene may be amplified. Many approaches are being taken to address the challenge presented by complex trait genetics, and data are being amassed at an alarming rate. Access to these data is crucial for coordination of efforts and avoidance of unnecessary duplication of research. This appendix describes some of the resources available on the World Wide Web that provide genetic and genomic data, tools for analyzing genome data, information on funding opportunities, and information about ethical, legal and social issues associated with the genetic analysis of disease traits.
Synthesis of N2‐Substituted Deoxyguanosine Nucleosides from 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine
Authors: Harris Thomas M. , Harris Constance M.
Published: June 2004

Syntheses of N2‐substituted nucleosides have been studied for many years, primarily with ribonucleosides. However, the primary route to these compounds requires acidic conditions that are too vigorous for the acid‐labile deoxyribonucleosides. The current strategy takes advantage of methods for low‐temperature, nonaqueous diazotization of ribosides in organic solvents using t‐butyl nitrate as the diazotizing agent and HF/pyridine as the fluoride source for the preaparation of a 2‐fluoro‐2?‐deoxyinosine derivative that can be used to synthesize N2‐substituted deoxyguanosine.
Synthesis, Characterization, and Application of Substituted Pyrazolopyrimidine Nucleosides
Authors: Dempcy Robert O. , Podyminogin Mikhail A. , Reed Michael W.
Published: June 2004

This unit describes, in detail, the preparation of 3‐aminopropyl‐substituted pyrazolo[3,4‐d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so‐called aminopropyl‐PPA and ‐PPG nucleosides (AP‐PPA and AP‐PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP‐PPA and AP‐PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl‐SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.
Nucleobase Protection of Deoxyribo‐ and Ribonucleosides
Authors: Iyer Radhakrishnan P.
Published: June 2004

Protecting groups for the imide/lactam function of thymine/uracil and guanine, respectively, prevent irreversible nucleobase modifications that may occur in the presence of alkylating or condensing reagents that are commonly used in nucleoside protection and oligonucleotide synthesis. This unit reviews these protecting groups, and also identifies protecting groups for the exocyclic amino function of cytosine, adenine, and guanine. The unit also explores recent trends in nucleobase protection that permit reliable oligonucleotide synthesis and removal of N‐protecting groups under very mild conditions.
Protection of 2′‐Hydroxy Functions of Ribonucleosides
Authors: Reese Colin B.
Published: June 2004

The main purpose of this article is to discuss 2′‐protection in the context of effective oligoribonucleotide synthesis. Emphasis is placed on the 2′‐protecting groups of choice in the synthesis of oligo‐and polyribonucleotides, and the requirements that a protective group must satisfy to become the 2′‐hydroxyl‐protecting group of choice. Finally, the unit discusses the issue of 2′‐O‐acyl and 2′‐O‐silyl group migration to the 3′‐hydroxy function of ribonucleosides during protection, along with the consequences of the conditions used for their removal on the stability of internucleotide linkages.
2′‐Hydroxyl‐Protecting Groups that are Either Photochemically Labile or Sensitive to Fluoride Ions
Authors: Miller Tod J. , Schwartz Miriam E. , Gough Geoffrey R.
Published: June 2004

Protected ribonucleotide monomers are more difficult to obtain than their 2′‐deoxy counterparts because of the need to protect the 2′‐hydroxy function. This unit describes the stepwise preparation of suitably 2′‐protected ribonucleosides using two protecting groups: 2‐nitrobenzyloxymethyl (NBOM) and tert‐butyldimethylsilyl (TBDMS). In addition, details are given for protecting the 5′‐hydroxyl and the nucleobase, yielding nucleosides that are easily converted to phosphoramidite or H‐phosphonate derivatives for automated oligoribonucleotide synthesis.
Preparation of 5′‐Silyl‐2′‐Orthoester Ribonucleosides for Use in Oligoribonucleotide Synthesis
Authors: Scaringe Stephen A. , Kitchen David , Kaiser Robert J. , Marshall William S.
Published: June 2004

The recent discovery that small interfering RNAs (siRNAs) induce gene suppression in mammalian cells has sparked tremendous interest in using siRNA‐based assays and high‐throughput screens to study gene function. As a result, research programs at leading academic and commercial institutions have become a substantial and rapidly growing market for synthetic RNA. Important considerations in synthesizing RNA for biological gene function studies are sequence integrity, purity, scalability, and resistance to nucleases; ease of chemical modification, deprotection, and handling; and cost. Of the well‐established RNA synthesis methods, 2′‐ACE chemistry is the only one that meets all of these criteria. 2′‐ACE technology employs a unique class of silyl ethers to protect the 5′‐hydroxyl, in combination with an acid‐labile orthoester protecting group on the 2′‐hydroxyl (2′‐ACE). 2′‐ACE‐protected phosphoramidite monomers are joined using standard solid‐phase technology to achieve RNA synthesis at efficiencies rivaling those for DNA. This unit describes the synthesis of standard 5′‐silyl‐2′‐ACE‐protected phosphoramidites.
Chemical Synthesis of RNA Sequences with 2′‐O‐[(Triisopropylsilyl)oxy]methyl‐protected Ribonucleoside Phosphoramidites
Authors: Pitsch Stefan , Weiss Patrick A.
Published: June 2004

The [(triisopropylsilyl)oxy]methyl (TOM) group is a useful protecting group for the 2′‐OH of ribonucleosides to be used for oligoribonucleotide synthesis by the phosphoramidite method. It is completely stable to all reaction conditions applied during assembly and the first deprotection step. It does not interfere with the coupling reaction and leads to very good coupling yields under DNA‐coupling conditions. The final cleavage occurs quantitatively without concomitant destruction of the RNA product. This unit describes the synthesis of TOM‐phosphoramidites from 2′‐O‐TOM‐5′‐O‐dimethoxytrityl‐N‐acetyl ribonucleosides, oligoribonucleotide assembly on an automated DNA synthesizer, and subsequent deprotection. Preparation of the TOM‐protected ribonucleosides is presented elsewhere in the series.
3‐(N‐tert‐Butylcarboxamido)‐1‐propyl and 4‐Oxopentyl Groups for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis
Authors: Wilk Andrzej , Chmielewski Marcin K. , Grajkowski Andrzej , Beaucage Serge L. , Phillips Lawrence R.
Published: June 2004

This unit provides procedures for the preparation of deoxyribonucleoside phosphoramidites and appropriate phosphordiamidite precursors with P(III) protecting groups different than the standard 2‐cyanoethyl group. Specifically, these phosphoramidites are functionalized with the 3‐(N‐tert‐butylcarboxamido)‐1‐propyl or 4‐oxopentyl groups. The usefulness of these novel deoxyribonucleoside phosphoramidites in the solid‐phase synthesis of a 20‐mer DNA oligonucleotide and its phosphorothioated analog is demonstrated. It is also shown that removal of the 3‐(N‐tert‐butylcarboxamido)‐1‐propyl phosphate/thiophosphate‐protecting group from these oligonucleotides is rapidly effected under thermolytic conditions at neutral pH, whereas the 4‐oxopentyl group is preferably removed by treatment with pressurized ammonia gas or concentrated ammonium hydroxide at ambient temperature. These detailed methods constitute an economical and alkylation‐free approach to large‐scale preparations of therapeutic oligonucleotides.
3′‐Modified Oligonucleotides and their Conjugates
Authors: Lyttle Matthew H.
Published: June 2004

Solid‐phase synthesis of 3′‐aminoalkyl, 3′‐thioalkyl, and 3′‐polyethyleneglycol are described. The first two are important as specific points of attachment for a large variety of reporter groups; the latter is important because of its increased cell membrane permeability, which aids in the development of antisense drugs. These protocols cover details of the synthetic organic chemistry needed to make each solid support, as well as DNA synthesis, workup, characterization, and conjugation.
Modification of the 5′ Terminus of Oligodeoxyribonucleotides for Conjugation with Ligands
Authors: Asseline Ulysse , Thuong Nguyen T.
Published: June 2004

Ligands can be introduced at the 5′ terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5′ terminus of the oligonucleotide. These are then reacted to give the ligand‐oligonucleotide conjugate. This unit describes the addition of carboxylated and aminoalkylated linkers, and phosphorothioate, phosphate, and masked thiol groups to the 5′ terminus of an oligonucleotide. The addition of linkers to ligands and the final reaction that produces the ligand‐conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite‐ or H‐phosphonate‐mediated oligonucleotide synthesis.
Solid‐Phase Synthesis of 2′‐Deoxy‐2′‐fluoro‐ β‐D‐Oligoarabinonucleotides (2′F‐ANA) and Their Phosphorothioate Derivatives
Authors: Viazovkina Ekaterina , Mangos Maria M. , Elzagheid Mohamed I. , Damha Masad J.
Published: June 2004

This unit describes the chemical synthesis of 2'‐deoxy‐2'‐fluoro‐b‐D‐oligoarabinonucleotides (2'F‐ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.
Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition
Authors: Leuck Michael , Wolter Andreas
Published: June 2004

The 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

5′‐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

A 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

One 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

This 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

This 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

In 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

The 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

This 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

Preparation 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

Despite 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

An 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.
Determination of Binding Thermodynamics
Authors: Dedon Peter C.
Published: June 2004

This unit serves as a starting point for exploring the thermodynamic properties of interactions between small molecules and DNA. It covers the determination of simple, apparent association/dissociation constants. The concentration of DNA‐bound ligand and free ligand are determined and a binding constant is extracted from these data. Data gathering and curve fitting are discussed.
A Competition Dialysis Assay for the Study of Structure‐Selective Ligand Binding to Nucleic Acids
Authors: Chaires Jonathan B.
Published: June 2004

Unique 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

This 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

MALDI‐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

Because 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

Synthetic 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

In 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

Capillary 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

This 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

Current 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

When 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

Carotid 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

This 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

The 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

This 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

Magnetic 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.