Oligonucleotide-modified Nuclotides

Modified nucleotides are interesting tools: They can alter the characteristics of DNA in many ways and enable the applicability of DNA in several innovative biotechnological applications.The first part of this thesis contributes to a better understanding of the acceptance and incorporation mechanism of modified nucleotides by DNA polymerases. Therefore modified nucleotides were successfully synthesized and incorporated into DNA by diverse DNA polymerases

Barcoded Nucleotides

DNA as an information storage system is simple and at the same time complex owing to the various different arrangements of the four natural nucleotides.[1] The DNA synthesis by DNA polymerases is intriguing, since these enzymes are able to catalyze the elongation of the primer strand by recognizing the DNA template and selecting the corresponding nucleotide.[1b, 2] This feature can be exploited to diversify the four-base-code by substitution of the natural substrates with modified analogues.[3] Nucleotide analogues equipped with various marker groups (e.g. dyes, tags, or spin labels[4])can be employed in DNA polymerase catalyzed reactions to increase the application scope of DNA (e.g. sequencing,structural characterization, and immobilization[4d, 5]). The “information” embedded in the marker groups allow conclusions to be drawn from the evaluation of the resulting signals. A significant gain in information would result by embedding a marker that exhibits the properties of a barcode. Typically, the barcode label bears no descriptive data but it consists of a series of signs which code for the deposited information (the term was used in other contexts with DNA before).[6] For universal adoption the barcode should be simple, affixed easily, and allow a reliable assignment of the deposited information. Oligodeoxynucleotides (ODNs) meet the requirements of a barcode label to a great extent, since they have a simple code and can be distinguished bycharacteristics such as self-assembly and hybridization specificity. For a simple introduction of these DNA barcode labels into DNA, an enzyme-mediated approach utilizing ODN-modified nucleotides would be desirable.[7] However, the acceptance of these modified nucleotides by DNA polymerases should be hampered by the steric demand of the ODN-modified nucleotides. Herein, we show that despitethe steric demand the enzymatic synthesis of barcoded DNA is feasible by using ODN-modified nucleoside triphosphatesthat are about 40-times larger than the natural nucleotides and longer than the diameter of a DNA polymerase (Figure 1A)

DNA polymerase-catalyzed incorporation of nucleotides modified with a G-quadruplex-derived DNAzyme

We show that nucleotides which are modified with a G-quadruplex-derived DNAzyme are substrates for DNA polymerases. Based on this finding we developed a naked-eye detection system that allows the detection of single nucleotide variations in DNA

Structures of KlenTaq DNA Polymerase caught while incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates

The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2′-deoxynucleoside-5′-O-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications

Structures of <i>KlenTaq</i> DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates

The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2′-deoxynucleoside-5′-<i>O</i>-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications

Ionomics

&lt;p&gt;Trace elements in their ionic form mediate biochemical reactions in human cells by acting as enzyme cofactors or centers for stabilizing protein structures. Deficit or accumulation of these substances lead to cell toxicity and severe diseases in humans and therefore, intracellular trace ion concentrations (i.e. the "ionome") must be tightly controlled. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine and quantify the intracellular trace ion concentrations. In ionomics assays HEK cells overexpressing a particular doxycycline-inducible SLC transporter were used to quantify the change of inorganic ions by ICP-MS upon cell lysis. ICP-MS based ionomics is rather a low throughput assay, as a significant volume of sample is required.&lt;/p&gt

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery