2-aminopurine Research Articles

2-Amino purine as a probe of DNA deformability in damaged DNA.

2-Amino purine (2AP) is an isomer of adenine (6-aminopurine) that, unlike the canonical DNA bases, is highly fluorescent when free in solution. The fluorescence quantum yield of 2AP in water is 0.68, and its fluorescence intensity decay is monoexponential with a single lifetime of 10-11 ns. When incorporated into DNA, 2AP fluorescence is quenched strongly by interactions with its neighboring bases. Inter-base interactions in duplex DNA give rise to a multiexponential decay that reflects the highly heterogeneous environment sensed by the probe. We used steady-state and time-resolved 2AP fluorescence to probe DNA dynamics around a lesion. In this work, we focused on uracil, a highly mutagenic and common lesion in DNA. In the cell, uracil is removed by Uracil DNA-glycosylase (UNG). UNG excision efficiency depends on DNA sequence, and this research is part of a broader study aimed to elucidate the underlying principles that dictate UNG substrate specificity. Duplex DNA substrates were prepared by annealing uracil-containing oligonucleotides with complementary strands containing 2AP opposite to the uracil. Fluorescence lifetimes were measured with 40 ps resolution, and results were analyzed together with the measured fluorescence quantum yields to calculate the fraction of ultra-quenched 2AP molecules. A higher fraction indicates a higher fraction of highly stacked (and therefore efficiently quenched) 2AP molecules, which we interpret as evidence for a less deformable substrate. This interpretation is supported by NMR imino proton exchange measurements and molecular dynamics simulations on the same substrates.

A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3′,3′-cGAMP

Cyclic dinucleotides have emerged as second messengers that regulate diverse processes in bacteria, as well as regulating the production of type I interferons in metazoans. Fluorescent sensors for these important second messengers are highly sought-after for high-throughput inhibitor discovery, yet most sensors reported to date are not amenable for high-throughput screening purposes. Herein, we demonstrate that a new analog, 3',3'-cG(d2AP)MP, which is a 2-aminopurine (2AP)-containing cyclic dinucleotide, self-associates in the presence of Mn(2+) with an association constant of 120,000 M(-1). 3'3'-cG(d2AP)MP can also form a heterodimer with cGAMP, activator of immune regulator, STING, or the bacterial biofilm regulator, c-di-GMP in the presence of Mn(II). Upon dimer formation, the fluorescence of 3',3'-cG(d2AP)MP is quenched and this provides a convenient method to monitor the enzymatic processing of both DGC and PDE enzymes, opening up several opportunities for the discovery of inhibitors of nucleotide signaling.

6-Oxo and 6-thio purine analogs as antimycobacterial agents

6-Oxo and 6-thio analogs of purine were prepared based on the initial activity screening of a small, diverse purine library against Mycobacterium tuberculosis (Mtb). Certain 6-oxo and 6-thio-substituted purine analogs described herein showed moderate to good inhibitory activity. N9-substitution apparently enhances the anti-mycobacterial activity in the purine series described herein. Several 2-amino and 2-chloro purine analogs were also synthesized that showed moderate inhibitory activity against Mtb.

A simple method for C-6 modification of guanine nucleosides

A facile method for the introduction of various substituents at the C-6 position of guanosine and 2'-deoxyguanosine is reported. In a simple, 1-step transformation, tert-butyldimethylsilyl protected guanosine and 2'-deoxyguanosine were converted to the O(6)-(benzotriazol-1-yl) derivatives via reaction with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 1,8-diazabicyclo[5.4.0]undec-7ene (DBU). The easily isolated, stable and storable, O(6)-(benzotriazol-1-yl) guanosine derivatives upon exposure to a range of nucleophiles, under appropriate conditions, led to the C-6 modified 2-amino purine nucleoside analogues in good yields.

Formation and Study of Electron Transfer of a Self-assembled Through Hydrogen Bonding 2-Amino Purine:C60-Uracil Conjugate

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DNA base flipping by a base pair-mimic nucleoside

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson–Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol−1 while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol−1, respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.

6-Dimethyl amino purine and 2-amino purine inhibit the induction of expression of milk protein genes by prolactin.

Two protein kinase-inhibitors, 6-dimethyl amino purine and 2-amino purine inhibited induction of beta-casein synthesis by prolactin when added to the culture medium of rabbit mammary explant and cells. The accumulation of the mRNA for alpha s1- and beta-caseins and for whey acidic protein did not take place in the presence of the inhibitors whereas beta-actin mRNA concentration was not altered. In the same experimental conditions, H7, an inhibitor of protein kinase C and, to a lower extent, of protein kinase A did not prevent prolactin from acting. These data suggest for the first time that specific protein kinases are involved in the transduction of the prolactin signal to milk protein genes.

The role of dam methylation in controlling gene expression

The E. coii gene dam encodes an adenine-specific methylase which converts all adenine residues in the sequence GATC into 6-methyl adenine, dam type methylases seem to be ubiquitous in Enterobacteriaceae and Haemophilus, since DNA from these strains cross hybridizes with the cloned dam gene and they contain DNA resistant to MboI cleavage, which only cleaves non-methylated DNA [1]. However, dam methylation is absent from most species of eubacteria not phylogenetically related to E. coli and presumably evolved late in prokaryotic evolution [2]. At least, one reason why people have become more aware of dam (and dcm) methylation in the last decade is because certain restriction enzymes are sensitive to DNA methylation and hence overlapping dam and restriction sites are not cleaved [3]. The primary phenotype of dam mutations is a nearly total loss of adenine methylation [4]. However, a small residual amount of adenine methylation appears to remain in dam strains even in strains carrying an insertion of Tn9 in the dam gene [5]. There are numerous secondary phenotypes associated with dam mutations and they are presumably due to the lack of adenine methylation. These secondary phenotypes include increased sensitivity to UV and 2-amino purine, increasedspontaneous mutability, decreased stability of plasmids, especially those of the Rl family, increased spontaneous induction of 2 and increased recombination frequency [4, 6]. The presence of a plasmid carrying the wild type dam gene reverses these secondary phenotypes [7]. Second site mutations which suppress some of these secondary phenotypes (but not the primary defect, i.e., the lack of adenine methylation) have been isolated in the genes mutL, m a t h and routs [6]. The dam gene is not essential, as shown by the isolation of a Tn9 insertion in the gene [8], but dam mutations are not viable in combination with recA, lexA, recB, C or poiA mutations. These double mutants become viable, however, in the presence of mutL or routs alleles [9]. The inviability of double dam, rec mutations became more comprehensible with the observation that dam mutations result in enhanced expression of genes of the SOS regulon (i.e., recA, lexA, uvrA, uvrB, uvrD, sulA, dinA and dinF) [10], implying that a dam mutation results in a requirement for higher levels of these proteins. More recently, a third phenotype for the dam mutations has been described in the literature: that of controlling the level of expression of a miscellaneous selection of genes with GATC sequences in their promoter regions. It is this third phenotype which is the subject of this paper, but initially it is worth summarizing the evidence for the dam methylation affecting other phenot~,~,~ of the E. coli bacteria.

Incorporation of 2-Aminopurine into the Desoxyribonucleic Acid of Bacteria and Bacteriophages

In der vorliegenden Arbeit wird bewiesen, daß 2-Aminopurin in die Desoxyribonucleinsäure (DNS) von Bakterien (E. coli B-97) und Bakteriophagen (T4) inkorporiert wird. Beide Organismen wurden in Gegenwart von 3H-markiertem 2-Aminopurin gezüchtet, ihre DNS rein dargestellt, hydrolysiert und die Desoxyriboside (aus der Bakterien-DNS) und Basen (aus der Phagen-DNS) durch Säulenchromatographie getrennt. In beiden Hydrolysaten wurde das tritiierte 2-Aminopurin eindeutig als DNS-Bestandteil wiedergefunden. Die relative Häufigkeit des 2-Aminopurins bezüglich des Adenins war 1/3000 für die Phagen-DNS und 1/300 für die Bakterien-DNS. Diese Ergebnisse und die molekulare Struktur des 2-Aminopurins machen es sehr wahrscheinlich, daß seine hohe Mutagenität auf der Inkorporierung in die DNS beruht.