The electrophoretic mobility of DNA fragments differing by a single 3′‐terminal nucleotide in an automated capillary DNA sequencer

The electrophoretic mobility of DNA fragments that differ by a single 3′‐terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5′‐fluorescently labelled primer to generate DNA fragments with 3′‐hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3′‐terminal nucleotide. It was found that the 3′‐terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) –C > ?A > ?T > ?G (slower). The C‐terminated fragments were the fastest and the G‐terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of the Cy3 and Cy5 Fluorescent Labels to Protect a DNA Strand from Degradation under λ Exonuclease Treatment

λ Exonuclease hydrolyzes a 5′-phosphorylated strand of double-stranded DNA in the 5′–3′ direction. In this paper, the activity of the enzyme with respect to DNA substrates containing Cy3 and Cy5 fluorescent labels at their 5′ ends is studied for the first time. It is demonstrated using the fluorescence procedure for measuring the exonuclease activity that double-stranded DNA, of which both the 5′ ends contain these fluorophores, is not destroyed under the action of λ exonuclease. Using the electrophoretic separation of DNA in polyacrylamide gel, the process of obtaining single-stranded DNA from double-stranded precursors containing different labels at the 5′ ends is studied. It is shown that the introduction of Cy3 and Cy5 fluorophores to the 5′ end of the DNA strand protects this strand from enzyme damage both in the duplex and in single-stranded form, and these labels can be used to produce fluorescently labeled single-stranded DNA.     

Type I Photosensitization of 2′‐deoxyadenosine 5′‐monophosphate (5′‐dAMP) by Biopterin and its Photoproduct Formylpterin

Biopterin (Bip) and its photoproducts 6‐formylpterin (Fop) and 6‐carboxypterin (Cap) accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. These compounds absorb in the UV‐A inducing a potential photosensitizing action that can cause damage to DNA and other biomolecules. In this work, we have investigated the capability of these pterin derivatives (Pt) to act as photosensitizers under UV‐A irradiation for the degradation of 2′‐deoxyadenosine 5′‐monophosphate (5′‐dAMP) in aqueous solutions, as model DNA target. Steady‐state and time‐resolved experiments were performed and the effect of pH was evaluated. The results showed that photosensitized degradation of 5′‐dAMP was only observed under acidic conditions, and a mechanistic analysis revealed the participation of the triplet excited state of the pterin derivatives (³Pt*) by electron transfer yielding the corresponding pair of radical ions (Pt^?? and 5′‐dAMP^?+), with successive photosensitizer recovery by electron transfer from Pt^?? to O₂. Finally, 5′‐dAMP^?+ participates in subsequent reactions to yield degradation products.     

Synthesis of 3′‐Thioamido‐Modified 3′‐Deoxythymidine 5′‐Triphosphates by Regioselective Thionation and Their Use as Chain Terminators in DNA Sequencing

The thioamide derivatives 3′‐deoxy‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐[(2‐methyl‐1‐thioxopropyl)amino]thymidine ( 4a ) and 3′‐deoxy‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐{{6‐{[(9H‐(fluoren‐9‐ylmethoxy)carbonyl]amino}‐1‐thioxohexyl}amino}thymidine ( 4b ) were synthesized by regioselective thionation of the corresponding amides 3a and 3b with 2,4‐bis(4‐methoxyphenyl)‐1,3,2,4‐dithiadiphosphetane 2,4‐disulfide (Lawesson's reagent). The addition of exact amounts of pyridine to the reaction mixture proved to be essential for an efficient transformation. The thioamides were converted into the corresponding 5′‐triphosphates 6a and 6b . Compound 6a was chosen for DNA sequencing experiments, and 6b was further labelled with fluorescein (→ 8 ).     

Factors Affecting DNA–DNA Interstrand Cross‐Links in the Antiparallel 3′–3′ Sense: A Comparison with the 5′–5′ Directional Isomer

Reported herein is a study of the unusual 3′–3′ 1,4‐GG interstrand cross‐link (IXL) formation in duplex DNA by a series of polynuclear platinum anticancer complexes. To examine the effect of possible preassociation through charge and hydrogen‐bonding effects the closely related compounds [{trans‐PtCl(NH₃)₂}₂(μ‐trans‐Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂)]⁴⁺ (BBR3464, 1 ), [{trans‐PtCl(NH₃)₂}₂(μ‐NH₂(CH₂)₆NH₂)]²⁺ (BBR3005, 2 ), [{trans‐PtCl(NH₃)₂}₂(μ‐H₂N(CH₂)₃NH₂(CH₂)₄)]³⁺ (BBR3571, 3 ) and [{trans‐PtCl(NH₃)₂}₂{μ‐H₂N(CH₂)₃‐N(COCF₃)(CH₂)₄}]²⁺ (BBR3571‐COCF₃, 4 ) were studied. Two different molecular biology approaches were used to investigate the effect of DNA template upon IXL formation in synthetic 20‐base‐pair duplexes. In the “hybridisation directed” method the monofunctionally adducted top strands were hybridised with their complementary 5′‐end labelled strands; after 24 h the efficiency of interstrand cross‐linking in the 5′–5′ direction was slightly higher than in the 3′–3′ direction. The second method involved “postsynthetic modification” of the intact duplex; significantly less cross‐linking was observed, but again a slight preference for the 5′–5′ duplex was present. 2D [¹H, ¹⁵N] HSQC NMR spectroscopy studies of the reaction of [¹⁵N]‐ 1 with the sequence 5′‐d{TATACATGTATA}₂ allowed direct comparison of the stepwise formation of the 3′–3′ IXL with the previously studied 5′–5′ IXL on the analogous sequence 5′‐d(ATATGTACATAT)₂. Whereas the preassociation and aquation steps were similar, differences were evident at the monofunctional binding step. The reaction did not yield a single distinct 3′–3′ 1,4‐GG IXL, but numerous cross‐linked adducts formed. Similar results were found for the reaction with the dinuclear [¹⁵N]‐ 2 . Molecular dynamics simulations for the 3′–3′ IXLs formed by both 1 and 2 showed a highly distorted structure with evident fraying of the end base pairs and considerable widening of the minor groove.     

Synthesis and Some Biochemical Properties of a Novel 5,6,7,8‐Tetrahydropyrimido[4,5‐c]pyridazine Nucleoside

A novel nucleoside analogue is described based on the pyridazine ring system. The nucleoside was successfully incorporated into DNA via both its phosphoramidite and 5′‐triphosphate derivatives. Enzymatically, the analogue behaves essentially as thymidine: it is a good substrate for the DNA polymerases Taq and exonuclease‐free Klenow fragment, leading to full‐length products when present in either the primer or template strands. In hybridisation studies, the nucleoside displays ambiguous base‐pairing properties, including universal base properties.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.     

Label‐Free Quantification of 5‐Azacytidines Directly in the Genome

Azacytidines (AzaC and AzadC) are clinically relevant pharmaceuticals that operate at the epigenetic level. They are integrated into the genome as antimetabolites to block DNA methylation events. This leads to a reduction of the 5‐methyl‐2′‐deoxycytidine (m⁵dC) level in the genome, which can activate epigenetically silenced genes. Because of the inherent chemical instability of Aza(d)Cs, their incorporation levels in DNA and RNA are difficult to determine, which hinders correlation of therapeutic effects with incorporation and removal processes. Existing methods involve radioactive labeling and are therefore unsuitable to monitor levels from patients. We report here a new direct chemical method that allows absolute quantification of the levels of incorporated AzaC and AzadC in both RNA and DNA. Furthermore, it clarifies that Aza(d)C accumulates to high levels (up to 12.9 million bases per genome). Although RNA‐based antimetabolites are often 2′‐deoxygenated in vivo and incorporated into DNA, for AzaC we see only limited incorporation into DNA. It accumulates predominantly in RNA where it, however, only leads to insignificant demethylation.     

Flip‐type disorder in 3‐substituted 2,2′:5′,2′′‐terthiophenes

In the crystal structures of four thiophene derivatives, (E)‐3′‐[2‐(anthracen‐9‐yl)ethenyl]‐2,2′:5′,2′′‐terthiophene, C₂₈H₁₈S₃, (E)‐3′‐[2‐(1‐pyrenyl)ethenyl]‐2,2′:5′,2′′‐terthiophene, C₃₀H₁₈S₃, (E)‐3′‐[2‐(3,4‐dimethoxyphenyl)ethenyl]‐2,2′:5′,2′′‐terthiophene, C₂₂H₁₈O₂S₃, and (E,E)‐1,4‐bis[2‐(2,2′:5′,2′′‐terthiophen‐3′‐yl)ethenyl]‐2,5‐dimethoxybenzene, C₃₆H₂₆O₂S₆, at least one of the terminal thiophene rings is disordered and the disorder is of the flip type. The terthiophene fragments are far from being coplanar, contrary to terthiophene itself. The central C—C=C—C fragments are almost planar but the bond lengths suggest slight delocalization within this fragment. The crystal packing is determined by van der Waals interactions and some weak, relatively short, C—H...S and C—H...π directional contacts.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

Synthesis of DNA oligos containing 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytosine as hTDG inhibitor

As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA). Here, we present synthesis of DNA oligos containing a 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDG(cat), residues 111-308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDG(cat) to form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal details on how hTDG(cat) recognizes the DNA substrate.     

Synthesis and Characterization of New 5‐Linked Pinoresinol Lignin Models

Pinoresinol structures, featuring a β‐β′‐linkage between lignin monomer units, are important in softwood lignins and in dicots and monocots, particularly those that are downregulated in syringyl‐specific genes. Although readily detected by NMR spectroscopy, pinoresinol structures largely escaped detection by β‐ether‐cleaving degradation analyses presumably due to the presence of the linkages at the 5 positions, in 5‐5′‐ or 5‐O‐4′‐structures. In this study, which is aimed at helping better understand 5‐linked pinoresinol structures by providing the required data for NMR characterization, new lignin model compounds were synthesized through biomimetic peroxidase‐mediated oxidative coupling reactions between pre‐formed (free‐phenolic) coniferyl alcohol 5‐5′‐ or 5‐O‐4′‐linked dimers and a coniferyl alcohol monomer. It was found that such dimers containing free‐phenolic coniferyl alcohol moieties can cross‐couple with the coniferyl alcohol producing pinoresinol‐containing trimers (and higher oligomers) in addition to other homo‐ and cross‐coupled products. Eight new lignin model compounds were obtained and characterized by NMR spectroscopy, and one tentatively identified cross‐coupled β‐O‐4′‐product was formed from a coniferyl alcohol 5‐O‐4′‐linked dimer. It was demonstrated that the 5‐5′‐ and 5‐O‐4′‐linked pinoresinol structures could be readily differentiated by using heteronuclear multiple‐bond correlation (HMBC) NMR spectroscopy. With appropriate modification (etherification or acetylation) to the newly obtained model compounds, it would be possible to identify the 5‐5′‐ or 5‐O‐4′‐linked pinoresinol structures in softwood lignins by 2D HMBC NMR spectroscopic methods. Identification of the cross‐coupled dibenzodioxocin from a coniferyl alcohol 5‐5′‐linked moiety suggested that thioacidolysis or derivatization followed by reductive cleavage (DFRC) could be used to detect and identify whether the coniferyl alcohol itself undergoes 5‐5′‐cross‐linking during lignification.     

Synthesis of Photolabile 5′‐O‐Phosphoramidites for the Photolithographic Production of Microarrays of Inversely Oriented Oligonucleotides

The photolabile 3′‐O‐{[2‐(2‐nitrophenyl)propoxy]carbonyl}‐protected 5′‐phosphoramidites ( 16 – 18 ) were synthesized (see Scheme) for an alternative mode of light‐directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA synthesis occurred in 5′→3′ direction, in agreement with the orientation of enzymatic synthesis. Synthesis yields were as good as those of conventional reactions. The resulting oligonucleotides are attached to the surface via their 5′‐termini, while the 3′‐hydroxy groups are available as substrates for enzymatic reactions such as primer extension upon hybridization of a DNA template (see Fig. 2). The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA microarrays.     

DNA Binding and Cleavage Activity of Zinc(II) Complex of N,N′‐Bis(2‐guanidinoethyl)‐2,6‐pyridinedicarboxamide

The interaction of zinc(II) complex of N,N′‐bis(guanidinoethyl)‐2,6‐pyridinedicarboxamide (Gua) with DNA was studied by CD spectroscopy and agarose gel electrophoresis analysis. The results indicate that the DNA binding affinity of Zn²⁺‐Gua is stronger than that of Gua and the Zn²⁺‐Gua can promote the cleavage of phosphodiester bond of supercoiled DNA under a physiological condition, which is ～10⁶ times higher than DNA natural degradation. The hydrolysis pathway was proposed as the possible mechanism for DNA cleavage promoted by the Zn²⁺‐ Gua. The acceleration is due to cooperative catalysis of the zinc cation center and the functional groups (bisguanidinium groups).     

3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotide Duplexes (=p‐DNA Duplexes) as Substitutes of RNA Hairpin Structures

By automated synthesis, we prepared hybrid oligonucleotides consisting of covalently linked RNA and p‐DNA sequences (p‐DNA=3′‐deoxyribopyranose (4′→2′)‐oligonucleotides) (see Table 1). The pairing properties of corresponding hybrid duplexes, formed from fully complementary single strands were investigated. An uninterrupted π‐π‐stacking at the p‐DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4′‐p‐DNA‐2′→5′‐RNA‐3′ and 5′‐RNA‐2′→4′‐p‐DNA‐2′ phosphodiester linkage, respectively (see Fig. 4). The RNA 2′‐phosphoramidites 9 – 12 , required for the formation of the RNA‐2′→4′‐p‐DNA phosphodiester linkage were synthesized from the corresponding, 3′‐O‐tom‐protected ribonucleosides (tom=[(triisopropylsilyl)oxy]methyl; Scheme 1). Analogues of the flavin mononucleotide (=FMN) binding aptamer 22 and the hammerhead ribozyme 25 were prepared. Each of these analogues consisted of two p‐DNA/RNA hybrid single strands with complementary p‐DNA sequences, designed to substitute stem/loop and stem motifs within the parent compounds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p‐DNA/RNA hybrid sequences resulted in the formation of the fully functional analogues 23 ⋅ 24 and 27 ⋅ 28 of the FMN‐binding aptamer and of the hammerhead ribozyme, respectively.     

One‐Step Formation of “Chain‐Armor”‐Stabilized DNA Nanostructures

DNA‐based self‐assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA‐based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6‐helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3′‐ends and azides on their 5′‐ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so‐called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.     

Nucleotides,Part LXV,Synthesis of 2′‐Deoxyribonucleoside 5′‐Phosphoramidites: New Building Blocks for the Inverse (5′‐3′)‐Oligonucleotide Approach

The syntheses of the 3′‐O‐(4,4′‐dimethoxytrityl)‐protected 5′‐phosphoramidites 25 – 28 and 5′‐(hydrogen succinates) 29 – 32 , which can be used as monomeric building blocks for the inverse (5′‐3′)‐oligodeoxyribonucleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two slightly different four‐step syntheses starting with the base‐protected nucleosides 13 – 20 . For the protection of the aglycon residues, the well‐established 2‐(4‐nitrophenyl)ethyl (npe) and [2‐(4‐nitrophenyl)ethoxy]carbonyl (npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of 3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator (instead of 1H‐tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the efficiency of this inverse strategy by the synthesis of the oligonucleotide 3′‐conjugates 33 and 34 which carry lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3′‐3′)‐ and (5′‐5′)‐internucleotide linkages (see Table 2).     

Fingerprinting DNA Oxidation Processes: IR Characterization of the 5‐Methyl‐2′‐Deoxycytidine Radical Cation

Methylated cytidine plays an important role as an epigenetic signal in gene regulation. Its oxidation products are assumed to be involved in active demethylation processes but also in damaging DNA. Here, we report the photochemical production of the 5‐methyl‐2′‐deoxycytidine radical cation via a two‐photon ionization process. The radical cation is detected by time‐resolved IR spectroscopy and identified by band assignment using density functional theory calculations. Two final oxidation products are characterized with liquid chromatography coupled to mass spectrometry.     

O6‐Alkylguanine DNA Alkyltransferase Repair Activity Towards Intrastrand Cross‐Linked DNA is Influenced by the Internucleotide Linkage

Oligonucleotides containing an alkylene intrastrand cross‐link (IaCL) between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines (dG) were prepared by solid‐phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B‐form DNA. Human O⁶‐alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O⁶‐alkylene linked IaCL lacking the 5′‐3′‐phosphodiester linkage between the connected 2′‐deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O⁶‐atom of dG is influenced by the backbone phosphodiester linkage between the cross‐linked residues.