RESUMO
Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a ß-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.
Assuntos
Reatores Biológicos/virologia , DNA de Cadeia Simples/biossíntese , Escherichia coli/metabolismo , Microbiologia Industrial/métodos , Bacteriófago M13/genética , Reatores Biológicos/economia , DNA de Cadeia Simples/isolamento & purificação , Escherichia coli/genética , Escherichia coli/virologia , Microbiologia Industrial/economia , Nanotecnologia/economia , Nanotecnologia/métodos , Plasmídeos/genéticaRESUMO
Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.
Assuntos
Actinas/metabolismo , Hipocampo/metabolismo , Modelos Biológicos , Imagem Molecular/métodos , Neurônios/citologia , Neurônios/metabolismo , Animais , Teorema de Bayes , Células Cultivadas , Simulação por Computador , Feminino , Células HeLa , Hipocampo/citologia , Humanos , Cadeias de Markov , Camundongos , MicroRNAs/metabolismo , Microscopia de Fluorescência/métodos , Modelos Estatísticos , Reconhecimento Automatizado de Padrão/métodos , Transporte Proteico/fisiologia , SoftwareRESUMO
A novel numerical method for determining the conformational structure of macromolecules is applied to idealized biomacromolecules in solution. The method computes effective inter-residue interaction potentials solely from the corresponding radial distribution functions, such as would be obtained from experimental data. The interaction potentials generate conformational ensembles that reproduce thermodynamic properties of the macromolecule (mean energy and heat capacity) in addition to the target radial distribution functions. As an evaluation of its utility in structure determination, we apply the method to a homopolymer and a heteropolymer model of a three-helix bundle protein [Zhou, Y.; Karplus, M. Proc Natl Acad Sci USA 1997, 94, 14429; Zhou, Y. et al. J Chem Phys 1997, 107, 10691] at various thermodynamic state points, including the ordered globule, disordered globule, and random coil states.