Modifikuotų pirimidino nukleotidų naudojimas oligonukleotidų sintezei

Nucleobase-modified nucleotides are widely used for a variety of purposes, where C5/C7 modified pyrimidines and purines are the main focus. However, a little attention has been paid to altering other positions of nucleobases such as C6/C8 or O4/N4. To increase the portfolio of accessible modified nucleotides not only the functional group should be the variable, but also the proper nucleobase position. Moreover, artificial nucleobases as well as nucleobase imitators bearing nucleotides should be explored more. Here, a set of novel modified pyrimidine nucleotides bearing N4, C5 or thio modifications as well as pyridone-based nucleobases was investigated. The application of these nucleotides for the template-independent DNA synthesis using terminal deoxynucleotidyl transferase was demonstrated. N4-acylated cytidine derivatives were also shown to be good substrates for the template-dependent DNA polymerases. N4-acyl-modified DNA was shown to be protected against the cleavage by the tested restriction endonucleases. In addition, N4-acyl-modified DNA can be demodified using an aqueous ethanolamine solution. Modified DNA bearing photoactive functional groups appeared to be suitable for the UV-induced covalent cross-linking with interacting proteins and a variety of solid supports. Therefore, novel modified pyrimidine nucleotides are proposed to expand the toolbox of nucleotide derivatives for the synthesis of functionalized DNA considerably

A versatile method for the UVA-induced cross-linking of acetophenone-or benzophenone-functionalized DNA

Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N-4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3'-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3'-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N-4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids

N-4-acyl-2 '-deoxycytidine-5 '-triphosphates for the enzymatic synthesis of modified DNA

A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N-4-acylated 2'-deoxycytidine nucleotides (dC(Acyl)). We find that a variety of family A and B DNA polymerases efficiently use dC(Acyl)TPs as substrates. In addition to the formation of complementary C-Acyl center dot G pair, a strong basepairing between N-4-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo-), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dC(Acyl)TPs but is prone to form C-Acyl center dot A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N-4-acylated-deoxycytidine nucleotides. These data reveal novel N-4-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization

Modified nucleotides as substrates of terminal deoxynucleotidyl transferase

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N-4-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3'-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3'-modification of ONs with various photo-and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures

Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2 ',3 '-dideoxynucleotides

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2′,3′-dideoxynucleotide (ddONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension

Engineering of a chromogenic enzyme screening system based on an auxiliary indole-3-carboxylic acid monooxygenase

Here, we present a proof-of-principle for a new high-throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme-coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro-chromogenic substrate, which is transformed by the target enzyme to indole-3-carboxylic acid. The later compound is converted to indoxyl by a newly identified indole-3-carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme-producing colonies turn blue. Two types of pro-chromogenic substrates have been tested. Indole-3-carboxaldehydes and the amides of indole-3-carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine-tuning of the pro-chromogenic substrate allows screening enzymes with the desired substrate specificity