Endless: A Purine‐Binding RNA Motif that Can Be Expressed in Cells

Abstract

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. In vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality in 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.

Noncovalent Spin‐Labeling of DNA and RNA Triplexes

“…Further, the individual strands of the triplex may be covalently linked to produce intramolecular folding motifs. The high affinity of such rationally designed DNA or RNA triplex motifs for purine nucleotides has been used to capture and release cofactors and to modulate the level of a second messenger in vivo . Reinforcing the gap with linkers can improve binding, and the binding capacity can be optimized by introducing several binding sites per triplex .…”

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures

“…Two extended hybrids were prepared that can bind with higher affinity to partner strands of a three‐dimensional network, expanding the scope of the methodology for assembling nanoporous materials . We also wished to create larger structures with several binding motifs for complexing adenine‐containing ligands in designed binding pockets, by a combination of Watson–Crick and Hoogsteen base pairing . In the event, we wished to create binding motifs with a high affinity for cofactors as ligands, by employing oligopurine chains as the middle strand of the triplex that feature an abasic site analog at the position where the adenine was to be bound by base pairing .…”

DNA Triplexes That Bind Several Cofactor Molecules

“…If so, they may solve practical problems in the realization of molecular machines based on biopolymers that performf unctions without the need for combustion or electricity.Thermal release of ligands mayalso be used to trigger reactions inside the body or to control the concentration of signaling molecules. [24] Ta ken together,o ur results confirmt hat DNA can act as as torage material, not just as genetic polymer or nanoscale buildingm aterial. (24), with the linking deoxycytidinen ucleotide (C*) showni nthe expansion box.…”

“…[16] Strands 1p and 1f featured three of the shorter linkers in ar ow to yield enlarged binding sites. The corresponding strand with ah exaethylene glycol linker 1h had been studied previously in adifferent context [24] and wasincluded in the current assays, but it was not synthesized again for the current work. Strands 4f-p with three isolated linker moieties were prepared with any of the three different linkers (f, h or p), www.chemeurj.org whereas the longesto ft he strands( 7f and 7p)w ere synthesized with the shorter linkers only,a ssuming thatt he h linker would reduce the cooperativity of triplex formation to an unacceptablee xtent.A ll modified strands were purified by reversed-phase HPLC, and were obtained in overall yields of 8-25 %, as calculated based on the loading of the solid support.…”

“…[23] Further,g ood binding selectivities can be achieved, [15] and intracellularl evels of purine nucleotides can be affected. [24] The molecular recognition throught wo-fold base pairing and stacking from either side is so extensive that it rivals that of binding pockets found in proteins binding adenosine triphosphate (ATP), [25] flavine adenined inucleotide (FAD), [26] 3',5'-cyclic adenosine monophosphate (cAMP), [27] or in aptamers [28][29] or riboswitches. [30][31] Triplexes are canonical structures in whichm ore than one binding site can be placed by straightforwardd esign.…”