RESUMEN
Invited for the cover of this issue are Sven Vollmer and Clemens Richert of the University of Stuttgart. The cover image hints at the analogy between a honey comb, as a macroscopic storage device, and DNA triplexes with designed binding sites, as molecular storage motifs that can release ATP to fuel a bioluminescence reaction. Read the full text of the article at 10.1002/chem.201503220.
Asunto(s)
Adenosina Trifosfato/química , ADN/química , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , Humanos , Mediciones Luminiscentes , Conformación de Ácido NucleicoRESUMEN
Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA.
Asunto(s)
Adenosina Trifosfato/química , ADN/síntesis química , Flavina-Adenina Dinucleótido/química , Oligonucleótidos/química , Purinas/química , Sitios de Unión , ADN/química , ADN/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , TermodinámicaRESUMEN
It is becoming increasingly clear that nature uses RNAs extensively for regulating vital functions of the cell, and short sequences are frequently used to suppress gene expression. However, controlling the concentration of small molecules intracellularly through designed RNA sequences that fold into ligand-binding structures is difficult. The development of "endless", a triplex-based folding motif that can be expressed in mammalian cells and binds the second messenger 3',5'-cyclic guanosine monophosphate (cGMP), is described. Inâ vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality inâ vivo, four endless RNA motifs arranged in tandem were co-expressed with a fluorescent cGMP sensor protein in murine vascular smooth muscle cells. Nitric oxide induced endogenous cGMP signals were suppressed in endless-expressing cells compared to cells expressing a control motif, which suggests that endless can act as a genetically encoded cGMP sink to modulate signal transduction in cells.