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.

No abstract

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 by characteristics 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 despite the steric demand the enzymatic synthesis of barcoded DNA is feasible by using ODN-modified nucleoside triphosphates that are about 40-times larger than the natural nucleotides and longer than the diameter of a DNA polymerase (Figure 1 A).Herein, we introduce 2'-deoxyribonucleotide analogues, containing an ODN at the nucleobase (Figure 1 B), as substrates for DNA polymerase mediated reactions. We chose the C5 position for pyrimidines and the C7 position for 7-deaza-purines to introduce the DNA strand at the nucleobase, since modifications at these positions have been accepted by DNA polymerases in several cases. [3,8] To ODNbarcode-label nucleotides, an ODN strand was activated with a commercial available carboxy modifier at the 5'-end while still on solid support and then coupled to the aminefunctionalized triphosphates (Figure 1 B, see Supporting Information). After deprotection and cleavage from the solid support, these ODN-functionalized nucleotides were tested in DNA polymerase promoted primer-extension reactions (yields and DNA sequences are listed in Figure 1 C and Supporting Information, Table S1). We examined the acceptance of the ODN-modified thymidine analogues by D...

We show that nucleotides which are modified with a G-quadruplexderived 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.Among the entirety of the human genome, single nucleotide polymorphisms (SNPs) are the most common variations.1 Between some of these dissimilarities and certain diseases, as well as direct effects of drugs on different patients, exists a direct linkage, a topic dealt with in pharmacogenomics. 1,2 For this reason, the development of methods that allow time-and cost-effective verification of distinct nucleotide variations in daily laboratory practice is important for the advance in personalized medicine. 3 Several assays were developed that allow the detection of single nucleotide variations during PCR from genomic DNA samples. Despite PCR-based technologies are broadly applied, they have the inherent disadvantage of requiring temperature cycling using sophisticated instrumentation. These disadvantages make PCR inappropriate for device portability, e.g. for point-of-care testing (POCT) or the detection of pathogens in the field. 4 To date, several naked-eye detection strategies have been exploited to identify single nucleotide variations and microbial pathogens, including urease/DNAzyme coupled system, 5 G-quadruplex DNAzyme biosensors, 6 and DNA probe-modified gold nanoparticles. 7 However, methods that link single nucleotide incorporation to a signal that is detectable by naked-eye are missing. Herein, we report a naked-eye detection system that converts the sequence-selective incorporation of a modified nucleotide into a colour change. The method is based on the introduction of a modified nucleotide that contains a G-quadruplex-derived DNAzyme by a DNA polymerase-mediated reaction. The approach is based on our recent finding that DNA polymerases are able to incorporate nucleotides that are modified with unstructured single stranded oligonucleotides. 8 Here we show that even nucleotides that bear a structured oligonucleotide ''cargo'' are sequence-selectively incorporated by DNA polymerases. Since the modification is a signaling unit, the sequence-selective incorporation can be coupled to a signal that is detectable by the naked eye. Our approach for the development of a DNA sequence selective naked-eye detection system is depicted in Fig. 1. It is based on performing primer extensions using a biotinylated primer immobilized on streptavidin-coated beads. Upon hybridisation to the appropriate target DNA template a DNAzyme-modified nucleotide will be sequence selectively incorporated. After washing steps, the addition of hemin, ABTS 2À , and H 2 O 2 will induce the formation of the biosensor with green colour generation. In order to build the depicted system, we first tethered the appropriate 25-nt long oligonucleotide that forms the DNAzyme to a nucleoside-5 0 O-triphosphate following the approach we reported recently. 8 We chose to functionalize the C5 position of the ...

Etiketten für DNA: Erstaunlicherweise sind DNA‐Polymerasen in der Lage, Oligodesoxynukleotid(ODN)‐modifizierte Nukleotide, die bis zu 40fach größer als ihre natürlichen Substrate sind, zu prozessieren, was zu Barcode‐markierter DNA führt (siehe Schema). Der Einbau der ODN‐modifizierten Nukleotide wurde in Lösung und an der festen Phase durch Hybridisierung mit einem fluoreszenzmarkierten DNA‐Strang komplementär zum ODN‐Strang nachgewiesen.