I‑Motif-Programmed Functionalization of DNA Nanocircles

The folding of various intra- and intermolecular i-motif DNAs is systematically studied to expand the toolbox for the control of mechanical operations in DNA nanoarchitectures. We analyzed i-motif DNAs with two C-tracts under acidic conditions by gel electrophoresis, circular dichroism, and thermal denaturation and show that their intra- versus intermolecular folding primarily depends on the length of the C-tracts. Two stretches of six or fewer C-residues favor the intermolecular folding of i-motifs, whereas longer C-tracts promote the formation of intramolecular i-motif structures with unusually high thermal stability. We then introduced intra- and intermolecular i-motifs formed by DNAs containing two C-tracts into single-stranded regions within otherwise double-stranded DNA nanocircles. By adjusting the length of C-tracts we can control the intra- and intermolecular folding of i-motif DNAs and achieve programmable functionalization of dsDNA nanocircles. Single-stranded gaps in the nanocircle that are functionalized with an intramolecular i-motif enable the reversible contraction and extension of the DNA circle, as monitored by fluorescence quenching. Thereby, the nanocircle behaves as a proton-fueled DNA prototype machine. In contrast, nanorings containing intermolecular i-motifs induce the assembly of defined multicomponent DNA architectures in response to proton-triggered predicted structural changes, such as dimerization, “kiss”, and cyclization. The resulting DNA nanostructures are verified by gel electrophoresis and visualized by atomic force microscopy, including different folding topologies of an intermolecular i-motif. The i-motif-functionalized DNA nanocircles may serve as a versatile tool for the formation of larger interlocked dsDNA nanostructures, like rotaxanes and catenanes, to achieve diverse mechanical operations

Allosteric Control of Oxidative Catalysis by a DNA Rotaxane Nanostructure

DNA is a versatile construction material for the bottom-up assembly of structures and functional devices in the nanoscale. Additionally, there are specific sequences called DNAzymes that can fold into tertiary structures that display catalytic activity. Here we report the design of an interlocked DNA nanostructure that is able to fine-tune the oxidative catalytic activity of a split DNAzyme in a highly controllable manner. As scaffold, we employed a double-stranded DNA rotaxane for its ability to undergo programmable and predictable conformational changes. Precise regulation of the DNAzyme’s oxidative catalysis can be achieved by external stimuli (i.e., addition of release oligos) that modify the spatial arrangement within the system, without interfering with the catalytic core, similar to structural rearrangements that occur in allosterically controlled enzymes. We show that multiple switching steps between the active and inactive conformations can be performed consistent with efficient regulation and robust control of the DNA nanostructure

Reversible Light Switch for Macrocycle Mobility in a DNA Rotaxane

A recent trend in DNA nanotechnology consists of the assembly of architectures with dynamic properties that can be regulated by employing external stimuli. Reversible processes are important for implementing molecular motion into DNA architectures as they allow for the regeneration of the original state. Here we describe two different approaches for the reversible switching of a double-stranded DNA rotaxane architecture from a stationary pseudorotaxane mode into a state with movable components. Both states only marginally differ in their respective topologies but their mechanical properties are fundamentally different. In the two approaches, the switching operation is based on strand-displacement reactions. One of them employs toehold-extended oligodeoxynucleotides whereas in the other one the switching is achieved by light-irradiation. In both cases, multiple back and forth switching between the stationary and the mobile states was achieved in nearly quantitative fashion. The ability to reversibly operate mechanical motion in an interlocked DNA nanostructure opens exciting new avenues in DNA nanotechnology

Input-Dependent Induction of Oligonucleotide Structural Motifs for Performing Molecular Logic

The K⁺–H⁺-triggered structural conversion of multiple nucleic acid helices involving duplexes, triplexes, G-quadruplexes, and i-motifs is studied by gel electrophoresis, circular dichroism, and thermal denaturation. We employ the structural interconversions for perfoming molecular logic operations, as verified by fluorimetry and colorimetry. Short G-rich and C-rich cDNA and RNA single strands are hybridized to produce four A-form and B-form duplexes. Addition of K⁺ triggers the unwinding of the duplexes by inducing the folding of G-rich strands into DNA- or RNA G-quadruplex mono- and multimers, respectively. We found a decrease in pH to have different consequences on the resulting structural output, depending on whether the C-rich strand is DNA or RNA: while the protonated C-rich DNA strand folds into at least two isomers of a stable i-motif structure, the protonated C-rich RNA strand binds a DNA/RNA hybrid duplex to form a Y·RY parallel triplex. When using K⁺ and H⁺ as external stimuli, or inputs, and the induced G-quadruplexes as reporters, these structural interconversions of nucleic acid helices can be employed for performing logic-gate operations. The signaling mode for detecting these conversions relies on complex formation between DNA or RNA G-quadruplexes (G4) and the cofactor hemin. The G4/hemin complexes catalyze the H₂O₂-mediated oxidation of peroxidase substrates, resulting in a fluorescence or color change. Depending on the nature of the respective peroxidase substrate, distinct output signals can be generated, allowing one to operate multiple logic gates such as NOR, INH, or AND

Reconfigurable Nanopolygons Made of DNA Catenanes

The development of new types of bonds and linkages that can reversibly tune the geometry and structural features of molecules is an elusive goal in chemistry. Herein, we report the use of catenated DNA structures as nanolinkages that can reversibly switch their angle and form different kinds of polygonal nanostructures. We designed a reconfigurable catenane that can self-assemble into a triangular or hexagonal structure upon addition of programmable DNA strands that function via toehold strand-displacement. The nanomechanical and structural features of these catenated nanojoints can be applied for the construction of dynamic systems such as molecular motors with switchable functionalities

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg²⁺ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron–electron double resonance spectroscopy (PELDOR or DEER) in the frozen state

SecinH3 reduces proliferation of lung cancer cells expressing wild-type EGFR.

<p>A, structures of the inhibitors used in this study. B – C, proliferation of A549 (B) and H460 (C) cells was determined by MTT assay in the presence of the indicated inhibitors. ***, p<0.001 relative to DMSO-treated cells.</p

Effect of gefitinib and SecinH3 on EGFR signaling.

<p>A – D, serum-starved cells were stimulated with EGF for 5 min and probed by western blot for the activation of the indicated proteins. A, representative Western blot. B – D, Statistical evaluation of the phosphorylation of EGFR (B), IRS1 (C), and Akt (D) in EGF-stimulated H460 cells in presence of gefitinib, SecinH3, or a combination of both inhbitors. p indicates the phosphorylated, i.e. activated, form of the protein. The signals were normalized for the loading control Hsc70. The error bars give the standard error. *, p<0.05, **, p<0.01, ***, p<0.001 relative to EGF-treated cells except for those comparisons which are specified by brackets.</p

SecinH3 retards growth of H460 xenografts in vivo.

<p>Mice bearing subcutaneous H460 xenografts were treated with SecinH3 by daily intraperitoneal injections. A, tumor volume was determined each day. On day 9 the experiment had to be stopped because the tumor volume in the control animals exceeded 1 cm³. B, distribution of tumor volumes on day 9. C – D, the degree of apoptosis was analyzed in sections of tumors on day 9. C, the number of fragmented nuclei was determined by TUNEL staining. D, cleaved, i.e. activated, caspase-3 was detected by imunohistochemistry. Representative tumor sections are shown. 5× and 40× indicate the magnification of the objective. For all graphs, means and standard errors are shown (n = 14). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Survivin and p27/Kip expression.

<p>A – C, H460 cells were treated overnight with 1 µM gefitinib, 15 µM SecinH3 or their combination. A, the expression of p27Kip and survivin was analyzed by western blot. B, C, Statistical evaluation of the western blot data shown in A. ** in B indicates p<0.01 relative to DMSO-treated controls as well as to gefitinib-treated cells. *** in C indicates p<0.001 relative to DMSO-treated controls. n = 3.</p