Nucleosides/nucleotides, labeling

Study Design Treatment, randomized, open label, active control, parallel assignment, safety/efficacy study Official Title A Phase III, Randomized, Open-Label Study of Lopina-vir/Ritonavir Tablets 800/200mg Once-Daily Versus 400/100mg Twice-Daily When Co-administered With Nucleoside/Nucleotide Reverse Transcriptase Inhibitors in Antiretroviral-Experienced, HIV-1 Infected Subjects Primary Outcome Measures ... 

Fischhaber, P. L., et al. (1997). Synthesis of duplex DNA containing a spin labeled analog of 2 -deoxycytidine. Nucleosides Nucleotides 16, 365-377. 

Nagahara, S., et ah (1992). Spin-labeled oligonucleotides site specifically labeled at the internucleotide linkage—Separation of stereoisomeric probes and EPR spectroscopical detection of hybrid formation in solution. Nucleosides Nucleotides 11, 889—901. 

M35. Muhlegger, K., Batz, H. G., Bohm, S., Eltz, H., Hditke, H. J., and Kessler, C. H., Synthesis and use of new digoxygenin-labeled nucleotides in non-radioactive labeling and detection of nucleic acids. Nucleosides Nucleotides 8, 1161-1163 (1988). 

Generally, all conversions in the biosynthetic direction, i.e. iPARMP— iPAR— iPA (catalysed by 5 -nucleotidase, (EC 3.1.3.5), and adenosine nucleosidase, (EC 3.2.2.7), respectively, c/. Fig. 2) may also proceed in the opposite direction, i.e. base-— nucleoside — nucleotide (catalysed by adenosine phosphorylase and adenosine kinase, respectively). All these enzymes require both Ade and iPA or Ado and iPAR, respectively, as substrates. They were characterised in wheat germ [15-18] and lupin seeds [19]. Interestingly, no K, -constants were reported for Z-type cytokinins (see summary in [22]). However, as seen in H-labelled Z-derivatives feeding experiments, Z-type cytokinins are also interconverted in a similar way [82,121,122]. Moreover, the specificity of these enzymes is not too strict with respect to the side chain configuration and one may speculate that this complex may function for most if not all native cytokinins [21,81]. 

DNA end. A slightly different type of PNA consists of amino adds with nudeobases in their side chains. PNA bind strongly to complementary DNA and RNA sequences. The high metabolic stability renders PNA interesting for in-vivo applications. However, further modifications are required in order to make it suitable for treatment in eukaryotic cells because of a lacking diffusion across lipid membranes. PNA may be used in diagnostic techniques for the detection of specific DNA, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA [T. Koch et al.. Nucleosides Nucleotides 1997,16, 1771 D. R. Corey, Trends Biotechnol. 1997, 15,224 E. Uhlmann, Biol. Chem. 1998,379,... 

Strube T, Schiemann O, MacMillan F, Prisner T, Engels JW (2001) A new facile method for spin-labeling of oligonucleotides. Nucleosides Nucleotides Nucleic Acids 20 (4-7) 1271-1274... 

Piton N, Schiemann O, Mu YG, Stock G, Prisner T, Engels JW (2005) Synthesis of spin-labeled RNAs for long range distance measurements by PELDOR. Nucleosides Nucleotides Nucleic Acids 24(5-7) 771-775... 

Kieper I, Schmidt T, FeraB, Ruterjans H (1988) N-labeled oligonucleotides - useful probes for H-NMR investigations. Nucleosides Nucleotides 7 821-825... 

De Vos, M -J, Cravador, A., Lenders, J.-P., Houard, S, and Bollen, A. (1990) Solid phase non isotopic labelling of oligodeoxynucleotides using 5 -protected aminoalkyl phosphoramidites. Application to the specific detection of human papilloma virus DNA Nucleosides Nucleotides 9, 259-273. 

Korlach, J. Bibillo, A. Wegener, J. Peluso, R Pham, X. X. Park, I. Clark, S. Otto, G. A. Xumer, S. W. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides, Nucleotides Nucleic Acids 2008,27,1072-1082. 

Patil, S. V. Salunkhe, M. M. Huorescent-labeled oligonucleotide probes with non-nucleotide linker detection of hybrid formation by fluorescence anisotropy. Nucleosides Nucleotides 1996, 15, 1603-1610. 

Schubert, F Cech, D. Wagner, K. Preparation of fluorophore derivatives for fluorescent labeling of nucleosides, nucleotides, and ohgonucleotides. Ger. Offen. DE 4023212,1991. 

Terminal transferase labeling was originally developed using radiolabeled (typically 32P) nucleoside triphosphates (Roychoudhury et al., 1979 Tu and Cohen, 1980). Later, the technique was extended to the use of nonradioactive nucleotide derivatives (Kumar et al., 1988). 

Regardless of the type of enzymatic labeling used, it is important that the label be incorporated into the nucleoside triphosphates or primers in a way that does not affect enzyme recognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA probes must start with chemical derivatization of individual nucleotides. Of the many chemical procedures that can be used to modify a nucleoside triphosphate monomer, there are only a few that will result in a derivative still able to be enzymatically added to an existing oligonucleotide strand. 

Some form of chemical labeling process must be used regardless of whether the final oligo conjugate is created by enzymatic or strictly chemical means. If enzymatic modification is to be done, the initial label still must be incorporated into an individual nucleoside triphosphate, which then is polymerized into an existing oligonucleotide strand (Section 1, this chapter). Fortunately, many useful modified nucleoside triphosphates are now available from commercial sources, often eliminating the need for custom derivatization of individual nucleotides. 

A typical experiment is sketched out in Fig. 3. A 32P-labeled nucleotide is used to measure the amount of newly synthesized RNA as a function of time. The concentrations of the substrate (the four nucleoside triphosphates of A, U, G, and C) and that of the enzyme are constant. The initial template concentration is varied by serial dilution with a constant dilution factor. The increase in RNA concentration in the course of time can be divided into three phases ... 

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