Funktionalisierte DNA: ein neues replizierbares Biopolymer

Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene‐ versus Alkyne‐Modified Nucleotides

“…Therefore, it is essential that the modification attached to the nucleotide does not significantly compromise enzyme activity. During the last years, constant efforts have been made to identify modified triphosphates (dN R TPs) that are well processed by DNA polymerases . Thereby nucleobase modifications are mainly attached at the C5 position of pyrimidines and the C7 position of 7‐deazapurines (Figure A) to direct the modifications pointing into the developing major groove of the DNA .…”

Generation and Enzymatic Amplification of High‐Density Functionalized DNA Double Strands

“…[5,6] Single-stranded highdensity functionalized DNA (fDNA) in which every base is modified with additional functionality can be generated by enzymatic primer extension of base-modified deoxynucleoside triphosphates (dNTPs) using conventional DNA as a template. [7,8] Single-stranded fDNAs containing residues 1-3 and 8 (Scheme 1) can in turn serve as templates in PCR reactions with natural nucleotides. [7] However, the utilization of a high-density fDNA template for the enzymatic synthesis of the corresponding fDNA counterstrand has so far been unsuccessful.…”

“…[7,8] Single-stranded fDNAs containing residues 1-3 and 8 (Scheme 1) can in turn serve as templates in PCR reactions with natural nucleotides. [7] However, the utilization of a high-density fDNA template for the enzymatic synthesis of the corresponding fDNA counterstrand has so far been unsuccessful. For this to occur, the polymerase would not only have to recognize the functionalized DNA strand as a template, but would also have to be able to incorporate modified bases-according to the directions of the templateopposite a certain base modification.…”

“…Herein we describe the first example of an enzymatically synthesized fDNA double strand in which all four nucleobases in each strand are substituted with various base analogues, thus allowing the introduction of up to eight different modifications into one dsDNA. The dNTP derivatives used (Scheme 1) cover a broad range of different functionalities, such as aromatic (3,7), basic (2, 8), acidic (1,5), and lipophilic residues (4, 6). [9] Furthermore, we have established a screen to identify and optimize conditions that enable the direct amplification of fully modified fDNA by PCR.…”

“…We synthesized a fully functionalized fDNA template enzymatically under similar conditions as described before from a 79 nucleotide 5'-biotinylated DNA template M79 using dNTPs 1-4. [7,10] The dNTPs 1-4 proved to be good substrates for the Vent(exo-), and Pwo DNA polymerase and replaced their natural counterparts sequence specifically (data not shown). We then tested whether fM79 could serve as a template in standard primer extensions in the presence of dNTPs 1-4 using Pwo DNA polymerase.…”