2,6-diaminopurine (Z)-containing toehold probes improve genotyping sensitivity.

2,6-diaminopurine (Z), a naturally occurring noncanonical nucleotide base found in bacteriophages, enhances DNA hybridization by forming three hydrogen bonds with thymine (T). These distinct biochemical characteristics make it particularly valuable in applications that rely on the thermodynamics of DNA hybridization. However, the practical use of Z-containing oligos is limited by their high production cost and the challenges associated with their synthesis. Here, we developed an efficient and cost-effective approach to synthesize Z-containing oligos of high quality based on an isothermal strand displacement reaction. These newly synthesized Z-oligos are then employed as toehold-blockers in an isothermal genotyping assay designed to detect rare single nucleotide variations (SNV). When compared with their counterparts containing the standard adenine (A) base, the Z-containing blockers significantly enhance the accuracy of identifying SNV. Overall, our innovative methodology in the synthesis of Z-containing oligos, which can also be used to incorporate other unconventional and unnatural bases into oligonucleotides, is anticipated to be adopted for diverse applications, including genotyping, biosensing, and gene therapy.

Use of 2,6-diaminopurine as a potent suppressor of UGA premature stop codons in cystic fibrosis.

Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.

Supramolecular Nature of Multicomponent Crystals Formed from 2,2'-Thiodiacetic Acid with 2,6-Diaminopurine or N9-(2-Hydroxyethyl)adenine.

The synthesis and characterization of the multicomponent crystals formed by 2,2'-thiodiacetic acid (H2tda) and 2,6-diaminopurine (Hdap) or N9-(2-hydroxyethyl)adenine (9heade) are detailed in this report. These crystals exist in a salt rather than a co-crystal form, as confirmed by single crystal X-ray diffractometry, which reflects their ionic nature. This analysis confirmed proton transfer from the 2,2'-thiodiacetic acid to the basic groups of the coformers. The new multicomponent crystals have molecular formulas [(H9heade+)(Htda-)] 1 and [(H2dap+)2(tda2-)]·2H2O 2. These were also characterized using FTIR, 1H and 13C NMR and mass spectroscopies, elemental analysis, and thermogravimetric/differential scanning calorimetry (TG/DSC) analyses. In the crystal packing the ions interact with each other via O-H⋯N, O-H⋯O, N-H⋯O, and N-H⋯N hydrogen bonds, generating cyclic hydrogen-bonded motifs with graph-set notation of R22(16), R22(10), R32(10), R33(10), R22(9), R32(8), and R42(8), to form different supramolecular homo- and hetero-synthons. In addition, in the crystal packing of 2, pairs of diaminopurinium ions display a strong anti-parallel π,π-stacking interaction, characterized by short inter-centroids and interplanar distances (3.39 and 3.24 Å, respectively) and a fairly tight angle (17.5°). These assemblies were further analyzed energetically using DFT calculations, MEP surface analysis, and QTAIM characterization.

Base-Pairing Properties of Double-Headed Nucleotides.

Nucleotides that contain two nucleobases (double-headed nucleotides) have the potential to condense the information of two separate nucleotides into one. This presupposes that both bases must successfully pair with a cognate strand. Here, double-headed nucleotides that feature cytosine, guanine, thymine, adenine, hypoxanthine, and diaminopurine linked to the C2'-position of an arabinose scaffold were developed and examined in full detail. These monomeric units were efficiently prepared by convergent synthesis and incorporated into DNA oligonucleotides by means of the automated phosphoramidite method. Their pairing efficiency was assessed by UV-based melting-temperature analysis in several contexts and extensive molecular dynamics studies. Altogether, the results show that these double-headed nucleotides have a well-defined structure and invariably behave as functional dinucleotide mimics in DNA duplexes.

The copious photochemistry of 2,6-diaminopurine: Luminescence, triplet population, and ground state recovery.

9H- and 7H-2,6-Diaminopurine (26DAP) photoinduced events in vacuum were studied at the MS-CASPT2/cc-pVDZ level of theory. The S1 1 (ππ* La ) state is initially populated evolving barrierless towards its minimum energy structure, from where two photochemical events can take place in both tautomers. The first is the return of the electronic population to the ground state via the C6 conical intersection (CI-C6). The second involves an internal conversion to the ground through the C2 conical intersection (CI-C2). According to our geodesic interpolated paths connecting the critical structures, the second route is less favorable in both tautomers, due to the presence of high energy barriers. Our calculations suggest a competition between fluorescence and ultrafast relaxation to the electronic ground state via internal conversion process. Based on our calculated potential energy surfaces and experimental excited state lifetimes from the literature, we can infer that the 7H- must have a greater fluorescence yield than the 9H-tautomer. We also explored the triplet state population mechanisms on the 7H-26DAP to understand their long-lived components observed experimentally.

A non-canonical nucleotide from viral genomes interferes with the oxidative DNA damage repair system.

Oxidative damage is a major source of genomic instability in all organisms with the aerobic metabolism. 8-Oxoguanine (8-oxoG), an abundant oxidized purine, is mutagenic and must be controlled by a dedicated DNA repair system (GO system) that prevents G:C→T:A transversions through an easily formed 8-oxoG:A mispair. In some forms, the GO system is present in nearly all cellular organisms. However, recent studies uncovered many instances of viruses possessing non-canonical nucleotides in their genomes. The features of genome damage and maintenance in such cases of alternative genetic chemistry remain barely explored. In particular, 2,6-diaminopurine (Z nucleotide) completely substitutes for A in the genomes of some bacteriophages, which have evolved pathways for dZTP synthesis and specialized polymerases that prefer dZTP over dATP. Here we address the ability of the GO system enzymes to cope with oxidative DNA damage in the presence of Z in DNA. DNA polymerases of two different structural families (Klenow fragment and RB69 polymerase) were able to incorporate dZMP opposite to 8-oxoG in the template, as well as 8-oxodGMP opposite to Z in the template. Fpg, a 8-oxoguanine-DNA glycosylase that discriminates against 8-oxoG:A mispairs, also did not remove 8-oxoG from 8-oxoG:Z mispairs. However, MutY, a DNA glycosylase that excises A from pairs with 8-oxoG, had a significantly lower activity on Z:8-oxoG mispairs. Similar preferences were observed for Fpg and MutY from different bacterial species (Escherichia coli, Staphylococcus aureus and Lactococcus lactis). Overall, the relaxed control of 8-oxoG in the presence of the Z nucleotide may be a source of additional mutagenesis in the genomes of bacteriophages or bacteria that have survived the viral invasion.

The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis.

Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.

Incorporation of Pseudo-complementary Bases 2,6-Diaminopurine and 2-Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization.

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.

Design and synthesis of a novel lanthanide fluorescent probe (TbIII-dtpa-bis(2,6-diaminopurine)) and its application to the detection of uric acid in urine sample.

In this study, a novel fluorescent probe, TbIII-dtpa-bis(2,6-diaminopurine) (Tb-dtpa-bdap), is designed based on the principle of complementary base pairing and synthesized for uric acid detection. The synthesized fluorescent probe is characterized by 1H NMR, 13C NMR, infra-red (IR) spectrum and ultraviolet-visible (UV-vis) spectra. It is found that the fluorescence of Tb-dtpa-bdap solution can be quenched obviously in the presence of uric acid. The affecting factors, including solution acidity, uric acid concentration and interfering substances, on the detection of uric acid using this probe are examined. Under optimized conditions, the fluorescence intensities of Tb-dtpa-bdap solution towards different uric acid concentrations show a linear response in the range from 1.00 × 10-5 mol·L-1 to 5.00 × 10-5 mol·L-1 with a linear correlation coefficient (R2) of 0.9877. And the obtained limit of detection (LOD) is about 5.80 × 10-6 mol·L-1, which is lower than the level of uric acid in actual urine. The mechanism on the detection of uric acid by using Tb-dtpa-bdap is inferred from the experimental results. The facts demonstrate that the proposed fluorescent probe can be successfully applied for the determination of uric acid in human urine samples.