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1.
Nat Commun ; 15(1): 883, 2024 Jan 29.
Article in English | MEDLINE | ID: mdl-38287055

ABSTRACT

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.


Subject(s)
Artificial Cells , DNA , DNA/genetics , Gene Regulatory Networks , Nanotechnology , RNA, Messenger/metabolism
2.
Microb Cell Fact ; 22(1): 244, 2023 Nov 30.
Article in English | MEDLINE | ID: mdl-38037098

ABSTRACT

Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the system's three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.


Subject(s)
Nanotechnology , Protein Biosynthesis , Protein Subunits , Nanotechnology/methods , Computer Simulation , Silicon
3.
J Am Chem Soc ; 145(17): 9729-9736, 2023 05 03.
Article in English | MEDLINE | ID: mdl-37071757

ABSTRACT

Site-specific recombination is a cellular process for the integration, inversion, and excision of DNA segments that could be tailored for memory transactions in artificial cells. Here, we demonstrate the compartmentalization of cascaded gene expression reactions in a DNA brush, starting from the cell-free synthesis of a unidirectional recombinase that exchanges information between two DNA molecules, leading to gene expression turn-on/turn-off. We show that recombination yield in the DNA brush was responsive to gene composition, density, and orientation, with kinetics faster than in a homogeneous dilute bulk solution reaction. Recombination yield scaled with a power law greater than 1 with respect to the fraction of recombining DNA polymers in a dense brush. The exponent approached either 1 or 2, depending on the intermolecular distance in the brush and the position of the recombination site along the DNA contour length, suggesting that a restricted-reach effect between the recombination sites governs the recombination yield. We further demonstrate the ability to encode the DNA recombinase in the same DNA brush with its substrate constructs, enabling multiple spatially resolved orthogonal recombination transactions within a common reaction volume. Our results highlight the DNA brush as a favorable compartment to study DNA recombination, with unique properties for encoding autonomous memory transactions in DNA-based artificial cells.


Subject(s)
Polymers , Recombination, Genetic , DNA/genetics , Molecular Conformation , Recombinases
4.
Methods Mol Biol ; 2433: 135-149, 2022.
Article in English | MEDLINE | ID: mdl-34985742

ABSTRACT

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.


Subject(s)
DNA , Polymers , DNA/genetics , Gene Expression , RNA , Ribosomes
5.
ACS Synth Biol ; 10(3): 609-619, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33595282

ABSTRACT

The design of artificial cell models based on minimal surface-bound transcription-translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription-translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.


Subject(s)
Protein Biosynthesis , Ribosomes/metabolism , Cell-Free System , DNA/chemistry , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Fluorescence Recovery After Photobleaching , Models, Biological , RNA, Messenger/metabolism , Ribosomes/chemistry
6.
Nat Commun ; 11(1): 5648, 2020 11 06.
Article in English | MEDLINE | ID: mdl-33159084

ABSTRACT

Building autonomous artificial cells capable of homeostasis requires regulatory networks to gather information and make decisions that take time and cost energy. Decisions based on few molecules may be inaccurate but are cheap and fast. Realizing decision-making with a few molecules in artificial cells has remained a challenge. Here, we show decision-making by a bistable gene network in artificial cells with constant protein turnover. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow decision-making to a fuzzy and rapid regime dominated by small-number fluctuations. Gene regulation was observed at lower DNA and protein concentrations than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. The high-copy regime was characterized by a sharp transition and hysteresis, whereas the low-copy limit showed strong fluctuations, state switching, and cellular individuality across the decision-making point. Our results demonstrate information processing with low-power consumption inside artificial cells.


Subject(s)
Artificial Cells/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Dosage , Gene Expression Regulation , Gene Regulatory Networks
7.
Nat Nanotechnol ; 15(9): 783-791, 2020 09.
Article in English | MEDLINE | ID: mdl-32690886

ABSTRACT

The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.


Subject(s)
Bacteriophage T4/genetics , Immobilized Nucleic Acids/genetics , Multiprotein Complexes/biosynthesis , Multiprotein Complexes/genetics , Protein Engineering/instrumentation , Protein Engineering/methods , Cell-Free System , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Equipment Design , Escherichia coli/genetics , Gene Silencing , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Silicon , Viral Proteins/genetics , Viral Proteins/metabolism
8.
Sci Adv ; 6(16): eaaz6020, 2020 04.
Article in English | MEDLINE | ID: mdl-32494616

ABSTRACT

Ribosome biogenesis is an efficient and complex assembly process that has not been reconstructed outside a living cell so far, yet is the most critical step for establishing a self-replicating artificial cell. We recreated the biogenesis of Escherichia coli's small ribosomal subunit by synthesizing and capturing all its ribosomal proteins and RNA on a chip. Surface confinement provided favorable conditions for autonomous stepwise assembly of new subunits, spatially segregated from original intact ribosomes. Our real-time fluorescence measurements revealed hierarchal assembly, cooperative interactions, unstable intermediates, and specific binding to large ribosomal subunits. Using only synthetic genes, our methodology is a crucial step toward creation of a self-replicating artificial cell and a general strategy for the mechanistic investigation of diverse multicomponent macromolecular machines.

9.
ACS Synth Biol ; 7(8): 1829-1833, 2018 08 17.
Article in English | MEDLINE | ID: mdl-30036485

ABSTRACT

Direct electric-field manipulation of gene expression reactions would simplify the design of biochemical networks by replacing complex biomolecular interactions with push-button operations. Here, we applied a localized electric field gradient at megahertz frequency to manipulate a cell-free gene-expression reaction in a DNA compartment on a chip. We broke the spatial symmetry of a homogeneous reaction in the compartment by creating a trap for macromolecules in a region of maximal field intensity localized 50 µm from immobilized DNA. Free of biochemical regulation, we demonstrated protein synthesis oscillations by on/off switching of the electric field. In response to the field, ribosomes, RNA polymerases, and nascent RNA and proteins accumulated in the trap, and were then depleted from the DNA region where gene expression occurred. The resulting reduction in the rate of protein synthesis recovered back to steady-state when the field was off. The combination of electric field with compartmentalized cell-free gene expression reactions creates a simple, label-free approach for controlling biomolecules in space and time, opening possibilities for hybrid biological systems with a bioelectronic interface based on minimal biological parts design.


Subject(s)
Electrochemical Techniques/methods , DNA/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Gene Expression , Oligonucleotide Array Sequence Analysis , RNA/metabolism , Ribosomes/metabolism
10.
Angew Chem Int Ed Engl ; 57(17): 4783-4786, 2018 04 16.
Article in English | MEDLINE | ID: mdl-29469991

ABSTRACT

Lithographic patterning of DNA molecules enables spatial organization of cell-free genetic circuits under well-controlled experimental conditions. Here, we present a biocompatible, DNA-based resist termed "Bephore", which is based on commercially available components and can be patterned by both photo- and electron-beam lithography. The patterning mechanism is based on cleavage of a chemically modified DNA hairpin by ultraviolet light or electrons, and a subsequent strand-displacement reaction. All steps are performed in aqueous solution and do not require chemical development of the resist, which makes the lithographic process robust and biocompatible. Bephore is well suited for multistep lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision. As an application, we demonstrate compartmentalized, on-chip gene expression from three sequentially immobilized DNA templates, leading to three spatially resolved protein-expression gradients.


Subject(s)
DNA Probes/chemistry , DNA/genetics , DNA/chemistry , Gene Expression , Oligonucleotide Array Sequence Analysis , Optical Imaging
11.
Curr Opin Chem Biol ; 40: 37-46, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28628855

ABSTRACT

Building biological systems outside the cell is an emerging interdisciplinary research field aimed to study design principles, and to emulate biological functions for technology. Reconstructing programmable cellular functions, from assembly of protein/nucleic-acid machines to spatially distributed systems, requires implementing minimal systems of molecular interactions encoded in genes, source-sink protein expression dynamics, and materials platforms for reaction-diffusion scenarios. Here, we first review how molecular turnover mechanisms, combined with nonlinear interactions and feedback in cell-free gene networks enable programmable dynamic expression patterns in various compartments. We then describe recent work on spatially distributed protein expression reactions. Finally, we discuss progress and challenges in the study of programmable protein/nucleic-acid complexes.


Subject(s)
Cell-Free System/metabolism , Gene Expression , Gene Regulatory Networks , Proteins/genetics , Animals , Diffusion , Humans , Multiprotein Complexes/analysis , Multiprotein Complexes/genetics , Protein Biosynthesis , Proteins/analysis , Transcription, Genetic
12.
Nat Nanotechnol ; 11(12): 1076-1081, 2016 12.
Article in English | MEDLINE | ID: mdl-27501315

ABSTRACT

DNA can be programmed to assemble into a variety of shapes and patterns on the nanoscale and can act as a template for hybrid nanostructures such as conducting wires, protein arrays and field-effect transistors. Current DNA nanostructures are typically in the sub-micrometre range, limited by the sequence space and length of the assembled strands. Here we show that on a patterned biochip, DNA chains collapse into one-dimensional (1D) fibres that are 20 nm wide and around 70 µm long, each comprising approximately 35 co-aligned chains at its cross-section. Electron beam writing on a photocleavable monolayer was used to immobilize and pattern the DNA molecules, which condense into 1D bundles in the presence of spermidine. DNA condensation can propagate and split at junctions, cross gaps and create domain walls between counterpropagating fronts. This system is inherently adept at solving probabilistic problems and was used to find the possible paths through a maze and to evaluate stochastic switching circuits. This technique could be used to propagate biological or ionic signals in combination with sequence-specific DNA nanotechnology or for gene expression in cell-free DNA compartments.


Subject(s)
DNA/chemistry , Nanostructures/chemistry , Nanotechnology/methods , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Nucleic Acid Conformation , Static Electricity
13.
Acc Chem Res ; 47(6): 1912-21, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24856257

ABSTRACT

CONSPECTUS: The expression of genes in a cell in response to external signals or internal programs occurs within an environment that is compartmentalized and dense. Reconstituting gene expression in man-made systems is relevant for the basic understanding of gene regulation, as well as for the development of applications in bio- and nanotechnology. DNA polymer brushes assembled on a surface emulate a dense cellular environment. In a regime of significant chain overlap, the highly charged nature of DNA, its entropic degrees of freedom, and its interaction with transcription/translation machinery lead to emergent collective biophysical and biochemical properties, which are summarized in this Account. First, we describe a single-step photolithographic biochip on which biomolecules can be immobilized. Then, we present the assembly of localized DNA brushes, a few kilo-base pairs long, with spatially varying density, reaching a DNA concentration of ∼10(7) base pairs/µm(3), which is comparable to the value in E. coli. We then summarize the response of brush height to changes in density and mono- and divalent ionic strength. The balance between entropic elasticity and swelling forces leads to a rich phase behavior. At no added salt, polymers are completely stretched due to the osmotic pressure of ions, and at high salt they assume a relaxed coil conformation. Midrange, the brush height scales with ratio of density and ionic strength to the third power, in agreement with the general theory of polyelectrolyte brushes. In response to trivalent cations, DNA brushes collapse into macroscopic dendritic condensates with hysteresis, coexistence, and a hierarchy of condensation with brush density. We next present an investigation of RNA transcription in the DNA brush. In general, the brush density entropically excludes macromolecules, depleting RNA polymerase concentration in the brush compared to the bulk, therefore reducing transcription rate. The orientation of transcription promoters with respect to the surface also affects the rate with a lower value for outward compared to inward transcription, likely due to local changes of RNA polymerase concentrations. We hypothesize that equalizing the macromolecular osmotic pressure between bulk and brush with the addition of inert macromolecules would overcome the entropic exclusion of DNA associated proteins, and lead to enhanced biochemical activity. Finally, we present protein synthesis cascades in DNA brushes patterned at close proximity, as a step toward biochemical signaling between brushes. Examining the synthesis of proteins polymerizing into crystalline tubes suggests that on-chip molecular traps serve as nucleation sites for protein assembly, thereby opening possibilities for reconstituting nanoscale protein assembly pathways.


Subject(s)
DNA/chemistry , Macromolecular Substances/chemistry , Biocompatible Materials , Biophysics , Cell-Free System , Dendrites/chemistry , Entropy , Escherichia coli , Osmolar Concentration , RNA/chemistry
14.
Article in English | MEDLINE | ID: mdl-23894031

ABSTRACT

Large macromolecular assemblies are widespread in all cell types with diverse structural and functional roles. Whether localized to membranes, nuclei, or cytoplasm, multimeric protein-nucleic acid complexes may be viewed as sophisticated nanomachines, an inspiration to chemical design. The formation of large biological assemblies follows a complex and hierarchical self-assembly process via ordered molecular recognition events. Serving a paradigm for biological assembly, extensive past studies of T4 bacteriophage and bacterial ribosomes by many groups have been revealing distinct design strategies, yet these two very different multimeric complexes share common mechanistic motifs. An emerging biochip approach highlights two conceptual notions to promote the study of assembly pathways: cell-free expression provides coupling between synthesis and assembly; surface anchoring allows high-resolution imaging of structural intermediates and opens up opportunities for rewiring a network by defining unnatural scaffolds for synthetic design applications.


Subject(s)
Cell-Free System , Microchip Analytical Procedures , Nanostructures , Nanotechnology , Proteins
15.
Nat Nanotechnol ; 7(6): 374-8, 2012 May 27.
Article in English | MEDLINE | ID: mdl-22635100

ABSTRACT

Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.


Subject(s)
Escherichia coli/chemistry , Lab-On-A-Chip Devices , Nanotubes, Peptide/chemistry , Protein Biosynthesis , Recombinant Proteins/biosynthesis , Silicon Dioxide/chemistry , Cell-Free System/chemistry , Recombinant Proteins/chemistry
16.
J Phys Chem B ; 114(43): 13897-903, 2010 Nov 04.
Article in English | MEDLINE | ID: mdl-20942452

ABSTRACT

The contribution of sequence elements of human telomere DNA to the interaction of DNA with electrons has been analyzed. By applying wavelength dependent low-energy photoelectron transmission and two-photon photoemission spectroscopy, we investigated the density of states of DNA oligomers with partial sequence elements of the human telomere assembled as monolayers on gold. The findings demonstrate the role of the resonance states in the DNA in accepting electrons and the effect of the sequence on these states. When guanine (G) bases are clustered together, the resonance negative ion state is stabilized, as compared to oligomers containing the same number of G bases but distributed within the sequence. The electron-capturing probability of the human telomere-like oligomer, a sequence with an additional single adenine (A) base adjacent to the G cluster, is dramatically enhanced compared to the other oligomers studied, most likely due to the enhancement of the density of states near the highest occupied molecular orbital.


Subject(s)
Electrons , Telomere/chemistry , Telomere/genetics , Adenine , Base Sequence , DNA/chemistry , DNA/genetics , Humans , Photoelectron Spectroscopy
17.
J Phys Chem B ; 114(25): 8581-4, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20527823

ABSTRACT

Toward the construction of double stranded DNA-based biosensors, packing of thiolated double-stranded DNA adsorbed on gold nanoparticles was observed to induce DNA denaturation. The denaturation was investigated as a function of DNA density, nanoparticle surface area, and DNA length. Direct correlation was found between DNA surface coverage and the denaturation. Denaturation occurred only at high densities of adsorbed DNA and was dependent on DNA length and therefore stability, providing guidelines for controlled adsorption of dsDNA on GNPs. Our results invoke a model in which the formation of a thiol-gold bond competes with the free energy associated with the denaturation of two DNA strands. Denaturation vacates space for additional molecules to bind through a thiol-gold bond.


Subject(s)
DNA/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Biosensing Techniques , Nucleic Acid Denaturation
18.
Proc Natl Acad Sci U S A ; 107(7): 2836-41, 2010 Feb 16.
Article in English | MEDLINE | ID: mdl-20133663

ABSTRACT

The coalescence of basic biochemical reactions into compartments is a major hallmark of a living cell. Using surface-bound DNA and a transcription reaction, we investigate the conditions for boundary-free compartmentalization. The DNA self-organizes into a dense and ordered phase with coding sequences aligned at well-defined distances and orientation relative to the surface, imposing directionality on transcription. Unique to the surface in comparison to dilute homogeneous DNA solution, the reaction slows down early, is inhibited with increased DNA density, is favorable for surface-oriented promoters, and is robust against DNA condensation. We interpret these results to suggest that macromolecules (RNA polymerase and RNA), but not solutes (ions and nucleotides), are partitioned between immobilized DNA and the reservoir. Without any physical barrier, a nonequilibrium directional DNA transaction forms macromolecular gradients that define a compartment, thus offering a boundary-free approach to the assembly of a synthetic cell.


Subject(s)
Cell Physiological Phenomena/physiology , DNA/metabolism , Transcription, Genetic/physiology , DNA-Directed RNA Polymerases/metabolism , Kinetics , Microscopy, Fluorescence , Protein Array Analysis , RNA/metabolism
19.
J Am Chem Soc ; 131(1): 89-95, 2009 Jan 14.
Article in English | MEDLINE | ID: mdl-19128174

ABSTRACT

8-Oxo-7,8-dihydroguanosine (8-oxoG) is among the most common forms of oxidative DNA damage found in human cells. The question of damage recognition by the repair machinery is a long standing one, and it is intriguing to suggest that the mechanism of efficiently locating damage within the entire genome might be related to modulations in the electronic properties of lesions compared to regular bases. Using laser-based methods combined with organizing various oligomers self-assembled monolayers on gold substrates, we show that indeed 8-oxoG has special electronic properties. By using oligomers containing 8-oxoG and guanine bases which were inserted in an all thymine sequences, we were able to determine the energy of the HOMO and LUMO states and the relative density of electronic states below the vacuum level. Specifically, it was found that when 8-oxoG is placed in the oligomer, the HOMO state is at higher energy than in the other oligomers studied. In contrast, the weakly mutagenic 8-oxo-7,8-dihydroadenosine (8-oxoA) has little or no effect on the electronic properties of DNA.


Subject(s)
DNA Damage , DNA/chemistry , Guanosine/analogs & derivatives , Electrons , Gold/chemistry , Guanine/chemistry , Guanosine/chemistry , Mass Spectrometry/methods , Models, Molecular , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Oxidation-Reduction , Spectrum Analysis/methods , Thymidine/chemistry
20.
Nano Lett ; 9(2): 909-13, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19170553

ABSTRACT

Dense brushes of linear DNA polymers are assembled on a biochip with approximately 30 nm between anchorage points, amounting to a few mega-base-pairs/microm(3). In bulk solution, a barrier incurs to conjugate more than two end-functionalized DNAs. However, such doublets bind the surface with almost equal efficiency to singlets, suggesting that extended brush buildup reduces the barrier. On-chip transcription reveals that doublets are roughly 2-fold inefficient compared to singlets, a manifestation of the interaction of the enzymatic machinery with the dense brush. Synthetic gene brushes made of DNA conjugates provide simple means to regulate expression on a chip.


Subject(s)
Genes, Synthetic/genetics , Oligonucleotide Array Sequence Analysis/methods , DNA/analysis , DNA/genetics , Transcription, Genetic/genetics
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