RESUMEN
Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37â °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.
Asunto(s)
Aductos de ADN/química , ADN/química , Formaldehído/química , ARN/química , Compuestos de Anilina/química , Catálisis , ADN/metabolismo , Aductos de ADN/análisis , ARN/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , ortoaminobenzoatos/químicaRESUMEN
N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m(6)A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m(6)A. We show that the enzyme can be used to locate and quantify m(6)A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m(6)A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m(6)A in mammalian 28S rRNA is nucleotide 4190.
Asunto(s)
Adenosina/análogos & derivados , ADN Polimerasa Dirigida por ADN/metabolismo , ARN/química , Adenosina/análisis , Adenosina/química , Secuencia de Bases , Humanos , Metilación , ARN Mensajero/química , Especificidad por Sustrato , Thermus thermophilus/enzimología , Timidina/químicaRESUMEN
A new C-nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent Cu(II)-mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal-metal distance falls into the range of previously reported values. Fluorescence studies with a donor-DNA-acceptor system indicate that photoinduced charge-transfer processes across these metal-ion-mediated base pairs in DNA occur more efficiently than over natural base pairs.
Asunto(s)
ADN/química , ADN/metabolismo , Hidroxiquinolinas/síntesis química , Metales/química , Nucleósidos/química , Emparejamiento Base , Cobre , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Electrones , Hidroxiquinolinas/químicaRESUMEN
A new donor-DNA-acceptor system has been synthesized containing Nile red-modified 2'-deoxyuridine as charge donor and 6-N,N-dimethylaminopyrene-modified 2'-deoxyuridine as acceptor to investigate the charge transfer in DNA duplexes using fluorescence spectroscopy and time-resolved femtosecond pump-probe techniques. Fluorescence quenching experiments revealed that the quenching efficiency of Nile red depends on two components: 1) the presence of a charge acceptor and 2) the number of intervening CG and AT base pairs between donor and acceptor. Surprisingly, the quenching efficiency of two base pairs (73% for CG and the same for AT) is higher than that for one base pair (68% for CG and 37% for AT), while at a separation of three base pairs less than 10% quenching is observed. A comparison with the results of time-resolved measurements revealed a correlation between quenching efficiency and the first ultrafast time constant suggesting that quenching proceeds via a charge transfer from the donor to the acceptor. All transients are satisfactorily described with two decays: a rapid charge transfer with 600 fs (â¼10(12) s(-1)) that depends strongly and in a non-linear fashion on the distance between donor and acceptor, and a slower time constant of a few picoseconds (â¼10(11) s(-1)) with weak distance dependence. A third time constant on a nanosecond time scale represents the fluorescence lifetime of the donor molecule. According to these results and time-dependent density functional theory (TDDFT) calculations a combination of single-step superexchange and multistep hopping mechanisms can be proposed for this short-range charge transfer. Furthermore, significantly less quenching efficiency and slower charge transfer rates at very short distances indicate that the direct interaction between donor and acceptor leads to a local structural distortion of DNA duplexes which may provide some uncertainty in identifying the charge transfer rates in short-range systems.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , ADN/metabolismo , Transporte de Electrón , Oxazinas/química , Teoría Cuántica , Espectrometría de Fluorescencia , Temperatura , Uridina/químicaRESUMEN
Uridine, adenosine, guanosine, and cytidine that carry a propargyl group attached to the 2'-oxygen were converted efficiently to the corresponding nucleoside triphosphates (pNTPs). Primer extension experiments revealed that pUTP, pATP, and pGTP can be successfully incorporated in oligonucleotides in the so-called 9°N and Therminator DNA polymerases. Most importantly, the ethynyl group as single 2'-modification of the enzymatically prepared oligonucleotides can be applied for postsynthetic labeling. This was representatively shown by PAGE analysis after the "click"-type cycloaddition with the fluorescent nile red azide. These results show that the 2'-position as one of the most important modification sites in oligonucleotides is now accessible not only for synthetic, but also for enzymatic oligonucleotide preparation.
Asunto(s)
Adenosina Trifosfato/síntesis química , Química Clic/métodos , ADN/síntesis química , Guanosina Trifosfato/síntesis química , Oxazinas/síntesis química , Uridina Trifosfato/síntesis química , Adenosina Trifosfato/genética , ADN/genética , Colorantes Fluorescentes/síntesis química , Guanosina Trifosfato/genética , Oligonucleótidos/síntesis química , Oligonucleótidos/genética , Uridina Trifosfato/genéticaRESUMEN
The terpyridine ligand directly attached to the 5-position of a uridine allows metal-mediated DNA assembly towards potentially electronically coupled DNA conjugates.
Asunto(s)
ADN/química , Metales/química , Piridinas/química , Electroforesis en Gel de Poliacrilamida , Fluorescencia , LigandosRESUMEN
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as a fluorescent label can be incorporated into DNA by two conceptually different ways: the non-nucleosidic DNA base surrogate Bo exhibits high brightness but no preferential base-pairing properties, whereas the BODIPY-modified uridine BodU has reduced quantum yields but shows preferred Watson-Crick base pairing with adenine.
Asunto(s)
Colorantes/química , ADN/química , Colorantes Fluorescentes/química , Emparejamiento Base , Compuestos de Boro , Espectrometría de FluorescenciaRESUMEN
A new type of BODIPY-modified uridines that contain the fluorophore attached to the 5-position of uridine via a short phenylene bridge have been prepared and characterized by methods of the optical spectroscopy and electrochemistry. The spectroscopic and redox properties can be manipulated by substituent and ligand exchange. After synthetic incorporation into DNA via phosphoramidite chemistry the canonical base pairing is maintained due to the rigid phenylene bridge. Moreover, primer extension experiments show that BODIPY-modified uridines are still recognized as thymidine derivatives by DNA-polymerases and adenine is inserted as the correct counter base.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Sondas de Ácido Nucleico/química , Uridina/análogos & derivados , ADN Polimerasa Dirigida por ADN/metabolismo , Oxidación-ReducciónRESUMEN
The chromophores pyrene and bordipyrromethenylbenzene directly linked to the 5-position of uridine are tolerated and recognized as thymine derivatives by DNA polymerases in primer extension experiments.