Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Mitochondrial Metabolomics Using High-Resolution Fourier-Transform Mass Spectrometry
High-resolution Fourier-transform mass spectrometry (FTMS) provides important advantages in studies of metabolism because more than half of common intermediary metabolites can be measured in 10 min with minimal pre-detector separation and without ion dissociation. This capability allows unprecedented opportunity to study complex metabolic systems, such as mitochondria. Analysis of mouse liver mitochondria using FTMS with liquid chromatography shows that sex and genotypic differences in mitochondrial metabolism can be readily distinguished. Additionally, differences in mitochondrial function are readily measured, and many o...
Source: Springer protocols feed by Biochemistry - January 1, 2014 Category: Biochemistry Source Type: news
LC-MS Profiling to Link Metabolic and Phenotypic Diversity in Plant Mapping Populations
We present a specialized protocol for large-scale targeted and untargeted metabolite profiling for samples from large plant mapping populations using both reversed-phase and aqueous normal-phase LC-MS. This methodology provides a fast and combined targeted/nontargeted workflow as a powerful tool to discriminate related plant phenotypes and describes methods to combine mass features and agronomic traits to link phenotypic to metabolic traits independent of putative metabolite identities. This easily reproducible analytical strategy, in combination with a sophisticated data processing and analysis workflow, can be applicable...
Source: Springer protocols feed by Biochemistry - January 1, 2014 Category: Biochemistry Source Type: news
Global Metabolic Profiling Using Ultra-Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry
Currently, liquid chromatography-mass spectrometry (LC-MS) is one of the most important analytical technologies for detecting hundreds of metabolites in the field of metabolomics. A recent advance in LC that has impacted metabolomics is the development of UPLC (ultra-performance liquid chromatography). In this chapter, we describe the analytical methodologies for the global metabolic profiling of serum, urine, and tissue samples using UPLC-Q-TOF (quadrupole-time-of-flight)-MS. Aqueous metabolites are extracted after adding methanol/acetonitrile/acetone and then analyzed by UPLC-MS under positive and/or negative ionization ...
Source: Springer protocols feed by Biochemistry - January 1, 2014 Category: Biochemistry Source Type: news
Overview of Mass Spectrometry-Based Metabolomics: Opportunities and Challenges
The field of metabolomics has witnessed an exponential growth in the last decade driven by important applications spanning a wide range of areas in the basic and life sciences and beyond. Mass spectrometry in combination with chromatography and nuclear magnetic resonance are the two major analytical avenues for the analysis of metabolic species in complex biological mixtures. Owing to its inherent significantly higher sensitivity and fast data acquisition, MS plays an increasingly dominant role in the metabolomics field. Propelled by the need to develop simple methods to diagnose and manage the numerous and widespread huma...
Source: Springer protocols feed by Biochemistry - January 1, 2014 Category: Biochemistry Source Type: news
Site-Selective Scission of Human Genome Using PNA-Based Artificial Restriction DNA Cutter
Site-selective scission of genomes is quite important for future biotechnology. However, naturally occurring restriction enzymes cut these huge DNAs at too many sites and cannot be used for this purpose. Recently, we have developed a completely chemistry-based artificial restriction DNA cutter (ARCUT) by combining a pair of pseudo-complementary PNA (pcPNA) strands (sequence recognition moiety) and Ce(IV)/EDTA complex (molecular scissors). The scission site of ARCUT and its scission specificity can be freely modulated in terms of the sequences and lengths of the pcPNA strands so that even huge genomes can be selectively cut...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Assembly of PNA-Tagged Small Molecules, Peptides, and Carbohydrates onto DNA Templates: Programming the Combinatorial Pairing and Inter-ligand Distance
The biochemical stability and desirable hybridization properties of peptide nucleic acids (PNA) coupled to the robustness of the peptidic chemistry involved in their oligomerization make them an attractive nucleic acid tag to encode molecules and program their assembly into higher order oligomers. The ability to program the dimerization of ligands with controlled distance between the ligands has important applications in emulating multimeric interactions. Additionally, the ability to program different permutations of ligand assemblies in a combinatorial fashion provides access to a broad diversity and offers a rapid screen...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Sequence Selective Recognition of Double-Stranded RNA Using Triple Helix-Forming Peptide Nucleic Acids
Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail. ...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Formation and Characterization of PNA-Containing Heteroquadruplexes
The guanine quadruplex is a secondary structure formed by DNA and RNA that has been implicated in regulation of gene expression and maintenance of genome stability. Guanine-rich PNA oligomers can invade DNA or RNA quadruplex targets to form heteroquadruplex structures. Affinities in the low nanomolar range are routinely observed, making PNAs among the tightest binding of all quadruplex-targeted agents. Although inherently more promiscuous than heteroduplex formation based on Watson–Crick pairing, selectivity of heteroquadruplex formation can be improved through rational design of the sequence and backbone structure o...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Preparation of Metal-Containing Peptide Nucleic Acid Bioconjugates on the Solid Phase
Peptide nucleic acids (PNAs) are a class of artificial DNA/RNA analogues that have unique physicochemical properties, which include a high chemical stability, resistance to nucleases and proteases, and higher mismatch sensitivity than DNA. PNAs were initially anticipated to be useful for application in antisense and antigene therapies; however, their poor cellular uptake has limited their use for such purposes in the “real world.” Recently, it has been shown that the addition of metal complexes to these oligonucleotide analogues could open up new avenues for their utilization in various research fields. Such me...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Synthesis, Characterization, and Evaluation of Radiometal-Containing Peptide Nucleic Acids
Peptide nucleic acids (PNAs) have very attractive properties for applications in nuclear medicine. Because PNAs have high selectivity for DNA/RNA recognition, resistance to nuclease/protease degradation, and high thermal and radiolytic stabilities, PNA bioconjugates could transform the areas of diagnostic and therapeutic nuclear medicine. In this book chapter, we report on the current developments towards the preparation of radiometal-containing PNA constructs and summarize the protocols for labeling these probes with 99mTc, 111In, 64Cu, 90Y, and 177Lu. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Chiral PNAs with Constrained Open-Chain Backbones
Chiral open-chain PNAs have been shown to have improved properties in terms of control of helical handedness, DNA affinity, sequence selectivity, and cellular uptake. They can be synthesized either using preformed chiral monomers or by means of a submonomeric strategy. The former is preferred when only a stereogenic center is present at C-5, whereas for PNA-bearing substituents at C-2, the submonomeric approach is preferred, since racemization, generally occurring during the solid-phase synthesis, can be minimized by this procedure. Here we describe the protocols for the synthesis of PNA oligomers containing C-2- or C-5- (...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Cyclopentane Peptide Nucleic Acids
Incorporating a cyclopentane ring into the two-carbon unit of a peptide nucleic acid backbone increases its binding affinity to complementary nucleic acid sequences. This approach is a general method to improve binding and can be applied at either purine or pyrimidine bases. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
MiniPEG-γPNA
Peptide nucleic acids (PNAs) are attractive, as compared to other classes of oligonucleotides that have been developed to date, in that they are relatively easy to synthesize and modify, hybridize to DNA and RNA with high affinity and sequence selectivity, and are resistant to enzymatic degradation by proteases and nucleases; however, the downside is that they are only moderately soluble in aqueous solution. Herein we describe the protocols for synthesizing the second-generation γPNAs, both the monomers and oligomers, containing MiniPEG side chain with considerable improvements in water solubility, biocompatibility, ...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Antisense Effects of PNAs in Bacteria
Peptide nucleic acids (PNAs) are a class of artificial DNA/RNA analogues that have unique physicochemical properties, which include a high chemical stability, resistance to nucleases and proteases and higher mismatch sensitivity than DNA. PNAs were initially anticipated to be useful for application in antisense and antigene therapies; however, their poor cellular uptake has limited their use for such purposes in the “real world”. Recently, it has been shown that the addition of metal complexes to these oligonucleotide analogues could open up new avenues for their utilization in various research fields. Such met...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
Peptide Nucleic Acid-Mediated Recombination for Targeted Genomic Repair and Modification
The ability to directly manipulate the human genome to correct a disease-related mutation, introduce a sequence change that would lead to site-specific gene knockout, or increase gene expression is a very powerful tool with tremendous clinical value. Triplex formation by synthetic DNA-binding molecules such as peptide nucleic acids (PNAs) has been studied for over 20 years and much of the work in the last 10 years has shown its great promise in its use to direct site-specific gene modification for the use in gene therapy. In this chapter, detailed protocols are described for the design and use of triplex-forming PNAs to bi...
Source: Springer protocols feed by Biochemistry - December 4, 2013 Category: Biochemistry Source Type: news
