BioWires: Conductive DNA Nanowires in a Computationally-Optimized, Synthetic Biological Platform for Nanoelectronic Fabrication

DNA is an ideal template for a biological nanowire-it has a linear structure several atoms thick; it possesses addressable nucleobase geometry that can be precisely defined; and it is massively scalable into branched networks. Until now, the drawback of DNA as a conducting nanowire been, simply put, its low conductance. To address this deficiency, we extensively characterize a chemical variant of canonical DNA that exploits the affinity of natural cytosine bases for silver ions. We successfully construct chains of single silver ions inside double-stranded DNA, confirm the basic dC-Ag+-dC bond geometry and kinetics, and show length-tunability dependent on mismatch distribution, ion availability and enzyme activity. An analysis of the absorbance spectra of natural DNA and silver-binding, poly-cytosine DNA demonstrates the heightened thermostability of the ion chain and its resistance to aqueous stresses such as precipitation, dialysis and forced reduction. These chemically critical traits lend themselves to an increase in electrical conductivity of over an order of magnitude for 11-base silver-paired duplexes over natural strands when assayed by STM break junction. We further construct and implement a genetic pathway in the E. coli bacterium for the biosynthesis of highly ionizable DNA sequences. Toward future circuits, we construct a model of transcription network architectures to determine the most efficient and robust connectivity for cell-based fabrication, and we perform sequence optimization with a genetic algorithm to identify oligonucleotides robust to changes in the base-pairing energy landscape. We propose that this system will serve as a synthetic biological fabrication platform for more complex DNA nanotechnology and nanoelectronics with applications to deep space and low resource environments

Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling

During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling

Construction and characterization of metal ion-containing DNA nanowires for synthetic biology and nanotechnology

DNA is an attractive candidate for integration into nanoelectronics as a biological nanowire due to its linear geometry, definable base sequence, easy, inexpensive and non-toxic replication and self-assembling properties. Recently we discovered that by intercalating Ag+ in polycytosine-mismatch oligonucleotides, the resulting C-Ag+-C duplexes are able to conduct charge efficiently. To map the functionality and biostability of this system, we built and characterized internally-functionalized DNA nanowires through non-canonical, Ag+-mediated base pairing in duplexes containing cytosine-cytosine mismatches. We assessed the thermal and chemical stability of ion-coordinated duplexes in aqueous solutions and conclude that the C-Ag+-C bond forms DNA duplexes with replicable geometry, predictable thermodynamics, and tunable length. We demonstrated continuous ion chain formation in oligonucleotides of 11-50 nucleotides (nt), and enzyme ligation of mixed strands up to six times that length. This construction is feasible without detectable silver nanocluster contaminants. Functional gene parts for the synthesis of DNA- and RNA-based, C-Ag+-C duplexes in a cell-free system have been constructed in an Escherichia coli expression plasmid and added to the open-source BioBrick Registry, paving the way to realizing the promise of inexpensive industrial production. With appropriate design constraints, this conductive variant of DNA demonstrates promise for use in synthetic biological constructs as a dynamic nucleic acid component and contributes molecular electronic functionality to DNA that is not already found in nature. We propose a viable route to fabricating stable DNA nanowires in cell-free and synthetic biological systems for the production of self-assembling nanoelectronic architectures.status: publishe

Role of the distal hydrogen-bonding network in regulating oxygen affinity in the truncated hemoglobin III from Campylobacter jejuni

Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues

Life on the edge: active microbial communities in the Kryos MgCl2-brine basin at very low water activity

The Kryos Basin is a deep-sea hypersaline anoxic basin (DHAB) located in the Eastern Mediterranean Sea (34.98°N 22.04°E). It is filled with brine of re-dissolved Messinian evaporites and is nearly saturated with MgCl2-equivalents, which makes this habitat extremely challenging for life. The strong density difference between the anoxic brine and the overlying oxic Mediterranean seawater impedes mixing, giving rise to a narrow chemocline. Here, we investigate the microbial community structure and activities across the seawater–brine interface using a combined biogeochemical, next-generation sequencing, and lipid biomarker approach. Within the interface, we detected fatty acids that were distinctly 13C-enriched when compared to other fatty acids. These likely originated from sulfide-oxidizing bacteria that fix carbon via the reverse tricarboxylic acid cycle. In the lower part of the interface, we also measured elevated rates of methane oxidation, probably mediated by aerobic methanotrophs under micro-oxic conditions. Sulfate reduction rates increased across the interface and were highest within the brine, providing first evidence that sulfate reducers (likely Desulfovermiculus and Desulfobacula) thrive in the Kryos Basin at a water activity of only ~0.4 Aw. Our results demonstrate that a highly specialized microbial community in the Kryos Basin has adapted to the poly-extreme conditions of a DHAB with nearly saturated MgCl2 brine, extending the known environmental range where microbial life can persist