Nucleic acid requirements

An understanding of a wide variety of phenomena concerning conformational stabilities and molecule-molecule association (protein-protein, protein-ligand, and protein-nucleic acid) requires consideration of solvation effects. In particular, a quantitative assessment of the relative contribution of hydrophobic and electrostatic interactions in macromolecular recognition is a problem of central importance in biology. 

The PPC allows the generation of NADPH reduction equivalents required for cell anabolism, and ribose 5-phosphate molecules for the synthesis of nucleic acids. Alternatively, ribose 5-phosphate can also be generated or transformed into fructose 6-phosphate or glyceraldehyde 3-phosphate, providing metabolic flexibility to the cell, in order to balance the fluxes through these pathways. The flux through the PPC is related to the nucleic acid requirements for DNA duplication or RNA transcription, and could probably be controlled by the cell cycle (Wagner, 1997). 

This behavior can be seen as complementary to another aspect of protein folding the withdrawal of hydrophobic side chains from solvent. The latter minimizes perturbation by burying those portions of the polypeptide for which water is the poorest solvent. The former minimizes perturbation of solvent by what remains exposed. Not all biological macromolecules show so small an effect. Nucleic acids require for their hydration about twice the amount of water required by globular proteins (for heat capacity measurements comparing protein and tRNA, see Rupley and Siemankowski, 1986). It may be signihcant that DNA, with an extensive hydration shell, undergoes facile hydration-dependent conformational transitions, which are not found for proteins. 

The assembly of biological molecules, including proteins and nucleic acids, requires the generation of appropriate starting materials. We have already considered the assembly of carbohydrates in regard to the Calvin cycle and the pentose phosphate pathway (Chapter 20). The present chapter and the next two examine the assembly of the other important building blocks—namely, amino acids, nucleotides, and lipids. 

Following an electrophoretic run, the band from the tracking dye is often the only visible band. The detection of separated proteins and nucleic acids requires subsequent treatment of the separation pattern for visualization. This treatment may be performed directly on the gel, or may require a blotting step in which the entire separation pattern is transferred onto a thin membrane material. The choice of detection method depends on the concentrations of analytes in the separated zones and whether recovery of the purified sample is required. 

Synthesis of a phosphodiester bond in nucleic acids requires energy input. As a result, the nucleoside monophosphates in nucleic acids are built up from hydrolysis of nucleoside triphosphates. Cleaving a pyrophosphate from a nucleoside triphosphate yields a nucleoside monophosphate and enough free energy to make the formation of polynucleoside monophosphates (i.e., polynucleotides) thermodynamically favorable. 

Enzyme-linked immimosorbent assays (ELISAs) are sensitive enough to detect relevant concentrations of small molecules and proteins. Their detection limits are inadequate for direct analysis of DNA in most cases. Eor this reason, it was not until the development of PCR that nucleic acid analysis became routine. Prior to PCR, the large quantities of nucleic acid required for hybridization precluded routine use and mandated the use of radioactive probes. After PCR, the abundance of amplified sequences allowed many methods of detection. In this environment, it was natural that immunochemical technologies first developed for immunoassay would be applied to analysis of amplified DNA. 

Nucleic acids are linear, chain-like macromolecules that were first isolated from cell nuclei. Hydrolysis of nucleic acids gives nucleotides, which are the building blocks of nucleic acids, just as amino acids are the building blocks of proteins. A complete description of the primary structure of a nucleic acid requires knowledge of its nucleotide sequence, which is comparable to knowing the amino acid sequence in a protein. 

QM calculations represent the leading tool to study intrinsic molecular interactions in nucleic acids, such as base stacking and base pairing. However, the QM data should not be overinterpreted and any extrapolation to nucleic acids requires proper consideration of the gas phase nature of QM calculations. In addition, in order to obtain meaningful QM data, basic methodological requirements must be fulfilled. These include, in addition to the obvious selection of appropriate level of calculations, very careful selection or determination of geometries, which is discussed in detail in this chapter. 

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