RESUMO
We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.
Assuntos
Cromossomos Artificiais Bacterianos/química , DNA/química , Nucleotídeos/química , Análise de Sequência de DNA/métodos , Animais , Cromatografia Líquida de Alta Pressão , Cromossomos Artificiais Bacterianos/genética , Simulação por Computador , Cães , Nucleotídeos/genética , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
We have explored a novel strategy for controlling the infectivity of adenoviral vectors. This strategy involves a method whereby the infectivity of adenoviral vectors is neutralized by treatment of viral particles with a water-soluble, photocleavable biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to 365 nm light induces a reversal of the neutralizing, chemical modification, resulting in restoration of infectivity to the viral vectors. The light-directed transduction of target cells by photoactivatable adenoviral vectors was demonstrated successfully both in vitro and in vivo. This photochemical infectivity trigger possesses great potential, both as a research tool and as a novel tactic for the delivery of gene-transfer agents, since the infectivity of adenoviral vectors can be controlled externally in a versatile manner.
Assuntos
Adenoviridae/patogenicidade , Biotina/química , Vetores Genéticos/efeitos da radiação , Virulência/efeitos da radiação , Adenoviridae/efeitos dos fármacos , Adenoviridae/efeitos da radiação , Animais , Biotina/síntese química , Biotinilação/métodos , Western Blotting/métodos , Sobrevivência Celular/efeitos da radiação , Cães , Expressão Gênica/genética , Vetores Genéticos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Fotoquímica/métodos , Fatores de Tempo , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
SIR2 is a key regulator of the aging process in many model organisms. The human ortholog SIRT1 plays a pivotal role in the regulation of cellular differentiation, metabolism, cell cycle, and apoptosis. SIRT1 is an NAD(+)-dependent deacetylase, and its enzymatic activity may be regulated by cellular energy. There is a growing number of known SIRT1 substrates that contain epsilon-acetyl lysine but for which no obvious consensus sequence has been defined. In this study, we developed a novel unbiased method to identify deacetylase sequence specificity using oriented peptide libraries containing acetylated lysine. Following incubation with SIRT1, the subset of deacetylated peptides was selectively captured using a photocleavable N-hydroxysuccinimide (NHS)-biotin linker and streptavidin beads and analyzed using mass spectrometry and Edman degradation. These studies revealed that substrate recognition by SIRT1 does not depend on the amino acid sequence proximate to the acetylated lysine. This result brings us one step closer to understanding how SIRT1 and possibly other protein deacetylases chose their substrate.