RESUMO
RNase H1 cleaves the RNA strand of RNA:DNA hybrids. Replacement of RNA 2'-hydroxyls by fluorine (FRNA) is commonly used to stabilize aptamers and siRNAs. However, FRNA:DNA hybrids fail to elicit RNase H activity. The underlying reasons are unclear, as 2'-OH groups are not directly involved in cleavage. We determined the crystal structure of Bacillus halodurans RNase H bound to a FRNA:DNA hybrid. The structure points to dynamic (slippage of the FRNA:DNA hybrid relative to the enzyme), geometric (different curvatures of FRNA:DNA and RNA:DNA hybrids), and electronic reasons (Mg(2+) absent from the active site of the FRNA:DNA complex) for the loss of RNaseH activity.
Assuntos
Bacillus/enzimologia , DNA/química , Flúor/química , Mimetismo Molecular , RNA/química , Ribonuclease H/química , Cristalografia por Raios X , Conformação ProteicaRESUMO
Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.
Assuntos
Clivagem do DNA , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/genética , Ácido Apurínico/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , DNA/metabolismoRESUMO
A new scaffold for studying photoinduced charge transfer has been constructed by connecting a [Ru(Bpy)3](2+) donor to a bis(8-hydroxyquinolinate)2 copper [CuQ2] acceptor through a peptide nucleic acid (PNA) bridge. The luminescence of the [Ru(Bpy)3](2+*) donor is quenched by electron transfer to the [CuQ2] acceptor. Photoluminescence studies of these donor-bridge-acceptor systems reveal a dependence of the charge transfer on the length and sequence of the PNA bridge and on the position of the donor and acceptor in the PNA. In cases where the [Ru(Bpy)3](2+) can access the π base stack at the terminus of the duplex, the luminescence decay is described well by a single exponential; but if the donor is sterically hindered from accessing the π base stack of the PNA duplex, a distribution of luminescence lifetimes for the donor [Ru(Bpy)3](2+*) is observed. Molecular dynamics simulations are used to explore the donor-PNA-acceptor structure and the resulting conformational distribution provides a possible explanation for the distribution of electron transfer rates.
Assuntos
2,2'-Dipiridil/análogos & derivados , Cobre/química , Compostos Organometálicos/química , Ácidos Nucleicos Peptídicos/química , Ácido Quinolínico/química , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/química , Elétrons , Luminescência , Medições Luminescentes , Simulação de Dinâmica Molecular , Estrutura Molecular , Compostos Organometálicos/síntese química , Ácidos Nucleicos Peptídicos/síntese química , Processos Fotoquímicos , Ácido Quinolínico/síntese química , Temperatura de TransiçãoRESUMO
Single-step nonadiabatic electron tunneling models are widely used to analyze electrochemical rates through self-assembled monolayer films (SAMs). For some systems, such as nucleic acids, long-range charge transfer can occur in a "hopping" regime that involves multiple charge transfer events and intermediate states. This report describes a three-step kinetic scheme to model charge transfer in this regime. Some of the features of the three-step model are probed experimentally by changing the chemical composition of the SAM. This work uses the three-step model and a temperature dependence of the charge transfer rate to extract the charge injection barrier for a SAM composed of a 10-mer peptide nucleic acid that operates in the hopping regime.
Assuntos
Elétrons , Modelos Químicos , Ácidos Nucleicos Peptídicos/química , Algoritmos , Simulação por Computador , Cinética , Modelos Genéticos , TemperaturaRESUMO
Substitution of a nucleobase pair with a pair of 1,2-hydroxypyridinone (1,2-HOPO) ligands in the center of a 10-base-pair peptide nucleic acid (PNA) duplex provides a strong binding site for Eu(III) as evidenced by UV thermal melting curves, UV titrations, and luminescence spectroscopy. Eu(III) excitation spectra and luminescence lifetime data are consistent with Eu(III) bound to both 1,2 HOPO ligands in a PNA-HOPO duplex as the major species present in solution.
Assuntos
Európio/química , Compostos Organometálicos/síntese química , Ácidos Nucleicos Peptídicos/química , Piridonas/química , Sítios de Ligação , Ligantes , Luminescência , Estrutura Molecular , Compostos Organometálicos/química , Espectrofotometria Ultravioleta , Temperatura de TransiçãoRESUMO
Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.
Assuntos
Glicina/química , Ácidos Nucleicos/química , Peptídeos/química , Transporte de Elétrons , Glicina/análogos & derivados , Estrutura MolecularRESUMO
We studied the charge transfer properties of bipyridine-modified peptide nucleic acid (PNA) in the absence and presence of Zn(II). Characterization of the PNA in solution showed that Zn(II) interacts with the bipyridine ligands, but the stability of the duplexes was not affected significantly by the binding of Zn(II). The charge transfer properties of these molecules were examined by electrochemistry for self-assembled monolayers of ferrocene-terminated PNAs and by conductive probe atomic force microscopy for cysteine-terminated PNAs. Both electrochemical and single molecular studies showed that the bipyridine modification and Zn(II) binding do not affect significantly the charge transfer of the PNA duplexes.