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.

Transient N4 -Acyl-DNA Protection against Cleavage by Restriction Endonucleases.

A set of five N4 -acyl-modified 2'-deoxycytidine 5'-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N4 -acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N4 -acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N4 modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N4 -modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.

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.

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 N4-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 N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

N4-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 N4-acylated 2'-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-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 dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-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 N4-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.