Nucleic acids transfer from

Not all the cellular DNA is in the nucleus some is found in the mitochondria. In addition, mitochondria contain RNA as well as several enzymes used for protein synthesis. Interestingly, mitochond-rial RNA and DNA bear a closer resemblance to the nucleic acid of bacterial cells than they do to animal cells. For example, the rather small DNA molecule of the mitochondrion is circular and does not form nucleosomes. Its information is contained in approximately 16,500 nucleotides that func-tion in the synthesis of two ribosomal and 22 transfer RNAs (tRNAs). In addition, mitochondrial DNA codes for the synthesis of 13 proteins, all components of the respiratory chain and the oxidative phosphorylation system. Still, mitochondrial DNA does not contain sufficient information for the synthesis of all mitochondrial proteins most are coded by nuclear genes. Most mitochondrial proteins are synthesized in the cytosol from nuclear-derived messenger RNAs (mRNAs) and then transported into the mito-chondria, where they contribute to both the structural and the functional elements of this organelle. Because mitochondria are inherited cytoplasmically, an individual does not necessarily receive mitochondrial nucleic acid equally from each parent. In fact, mito-chondria are inherited maternally. 

Normal cells also were found to contain src-like genes when their DNA was hybridized with labeled nucleic acid probes from the v-src gene. As with other cellular genes, the c-onc genes were interrupted with introns. v-Onc genes lack introns. Consequently, in the distant past c-onc genes must have been transferred to the retroviruses. If the transfer had been from the virus to the cell, c-onc genes probably would not contain introns. 

Natural viruses provide us with perfect demonstrations of how effective nucleic acid transfer into mammalian cells can proceed. The secret of their efficiency is their dynamic, bioresponsive behavior during delivery, which distinguishes them from classic synthetic nanoparticles. Thus, it has been tempting for us and many research colleagues [69, 92, 164, 188-194] to design nucleic acid nanoparticles with virus-like characteristics ( synthetic viruses ). 

The aim of the nucleic acid or gene transfer is the genetic modification of human somatic cells, either in the human body, that is, in vivo, or outside the human body, that is, ex vivo, in the latter case followed by transfer of the modified cells to the human body [4, 5]. The simplest case of genetic modification of a cell results from the addition of a therapeutic gene encompassed by an expression vector [6]. At least in theory, nucleic acid transfer may also be aimed at exchange of individual... 

From the economic point of view, procedures for the manufacture of therapeutic DNA must be scalable and efficient, and, at the same time, simple and robust. Manufacturing processes are as manifold as the gene transfer methods used in gene therapy. As an example, manufacture of plasmid DNA for naked nucleic acid transfer can be briefly described as follows [26]. The methods available for plasmid production today largely originate from lab procedures for the production of DNA for analytical... 

Numerous fluorescent solution and solid-phase assays have been described to measure the amount of amplification product generated in each reaction, but they can fail to discriminate amplified DNA of interest from nonspecific amplification products due to the missing final confirmation step. Some of these analyses rely on blotting techniques, which introduce another variable due to nucleic acid transfer efficiencies, while other assays have been developed to eliminate the need for gel electrophoresis, yet provide the requisite specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way to overcome the need for post-PCR analysis by electrophoresis. 

Quake and coworkers [16] developed a PDMS microfluidic device (shown in Fig. 4c) for nucleic acid purification from a small number of bacterial or mammalian cells. This multilayer device contained fluidic channels and a system of membrane-actuated pneumatic valves and pumps, which enabled precise control of buffers, lysis agents, and cell solution and also allowed for parallel processing. Bacterial cells, dilution buffer, and lysis buffer are first introduced into the chip and then transferred into the rotary mixer. Once mixed, the lysate is flushed over a DNA affinity column and drained. The DNA... 

Results in Table 6 suggest that the transfer of a nucleic acid base from the gas phase to aqueous solution is very favorable. However, it does not necessarily mean that nucleic acid bases are hydrophilic, since the widely spred concept of hydrophilicity/hydrophobicity results from the transfer between organic and aqueous environments and not from the transfer between gas phase and water. A clear insight into the problem can be gained by comparing... 

Transfer RNA (tRNA) Transfer RNAs are relatively small nucleic acids containing only about 70 nucleotides They get their name because they transfer ammo acids to the ribosome for incorporation into a polypeptide Although 20 ammo acids need to be transferred there are 50-60 tRNAs some of which transfer the same ammo acids Figure 28 11 shows the structure of phenylalanine tRNA (tRNA ) Like all tRNAs it IS composed of a single strand with a characteristic shape that results from the presence of paired bases m some regions and their absence m others... 

Schmidt J.G., Nielsen P.E., Orgel L. E. Information transfer from peptide nucleic acids to RNA by template-directed syntheses. Nucleic Acids Res. 1997 25 4797- 802. 

The scientific world was amazed to hear that David Lee, from the laboratory of Reza Ghadiri (Scripps Research Institute, La Jolla, California), had found a self-replicating peptide (Lee et al., 1996) there are analogies to the experiments with oligonucleotides (see Sect. 6.4). Lee was able to show that a certain peptide, containing 32 amino acids, can both function as a matrix and also support its own synthesis autocatalytically. The information transfer is clearly more complex than that involved in nucleic acid replication. In the case of this particular peptide, both the... 

Intracellular pathways after escape from the endolysosomal system into the cytosol are less clear. Obvious bottlenecks include, in the case of gene transfer (pDNA delivery), cytosolic transport to the perinuclear area, nuclear uptake, and nuclear presentation of the pDNA to the transcriptional machinery in bioactive form. In the case of siRNA (or mRNA and some other nucleic acids such as oligonucleotides), cytosolic accessibility for the required function is essential. Besides cytosolic transport [176, 177] and the nuclear import of large nucleic acid molecules [178-180], incorporation of functional nuclear import peptide domains has been evaluated [181-186]. Another bottleneck, nucleic acid unpackaging [187], i.e., partial or complete dissociation from the polymeric carrier, which is required for biological accessibility of the delivered nucleic acid, will be discussed in Sect. 3.3. 

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