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

ABSTRACT: 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.

SUBMITTER: Vecchioni S

PROVIDER: S-EPMC6502794 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The secondary structure of the synthetic 17-residue peptide, conantokin-G (con-G), a gamma-carboxyglutamate-containing marine cone snail neuroactive protein, is altered from a random conformation to one containing a very high level (>70%) of alpha-helix on binding of multivalent cations. The proportion of alpha-helix formed correlated well with the size of the cation and ranged from a low of approx. 7% with large cations, such as Ba2+, to more than 70% with smaller cations, such as Mn2+, Mg2+ and Zn2+. The valency of the multivalent cation was not as important, since tervalent lanthanides (Eu3+, Gd3+ and Tb3+) of ionic radius 106-109 pm induced similar levels (50-60%) of helix to those induced by Ca2+ and Cd2+ (ionic radii 109 and 114 pm respectively). Although the correlation was not as tight, smaller cations of the same valency allowed the helical transition to occur at lower concentrations than the larger cations. The spectroscopic and spectrometric properties of some of these cations permitted a more detailed analysis of the molecular nature of the cation-con-G binding. EPR-based titrations with Mn2+ provided a binding isotherm that was deconvoluted to a single class of 2-3 Mn2+ sites of average Kd 3.9 microM. This number of sites was similar to that for Ca2+ [Prorok, Warder, Blandl and Castellino (1996) Biochemistry 35, 16528-16534], but a much lower Kd was displayed with Mn2+. Determinations by 1H NMR of the longitudinal relaxation rates of the water protons in Mn2+/con-G solutions at different magnetic field strengths corresponding to the proton Langmuir frequencies of 24, 300 and 500 MHz permitted calculation of the hydration number of Mn2+ in the complex, which was found to be 1.0. This indicates that five of the six co-ordination sites of Mn2+ are occupied by peptide atoms, probably oxygens. Titrations of the changes in Tb3+ fluorescence as a result of its binding to con-G gave an EC50 of 58 microM, a value nearly identical with that obtained by titration of the change in helicity of the peptide as a function of Tb3+ concentration. This shows that the macroscopic binding of Tb3+ to con-G is directly responsible for the alteration in secondary structure of the peptide. Finally, Cd2+ was found to be an extremely suitable cation for an NMR-based investigation of the amino acid residues of apo-con-G that are perturbed by cation binding. In a limited example of the results of this study, it was discovered that originally equivalent CH2(delta) protons of Arg13 became distinctly magnetically non-equivalent in the Cd2+-bound helical form of con-G. This indicates that Arg13 is situated in the helix in such a way that the mobility of its side chain is highly restricted. In conclusion, the data show that a variety of multivalent cations with measurable spectroscopic and spectrometric properties interact similarly with con-G and generate extensive alpha-helical conformation in this peptide.

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

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