Bimolecular I-motif Research Articles

A Janus-like DNA probekit for simultaneously imaging the homo- and heterodimerization of HER2 and HER3 on living tumor cells

HER2:HER2 homodimer and HER2:HER3 heterodimer are crucial participants and complicatedly interrelated in tumor progression and therapy. Here, we construct a Janus-like DNA probekit for simultaneously monitoring them on tumor cells. In this probekit, HER2 aptamer with a cytosine-rich stretch extended (Apt2-C) is labeled with FRET donor AF488 and acceptor AF594 at a distance apart; HER3 aptamer with a guanine-rich stretch extended (Apt3-G) is labeled with another FRET acceptor Cy3. Apt2-C and Apt3-G bind to HER2 and HER3 via their aptamers, respectively. Along with homodimerized HER2, two cytosine-rich stretches within Apt2-C are nearer and fold into a bimolecular i-motif, narrowing AF488 and AF594 to germinate FRET for homodimerization indication. Upon HER2:HER3 heterodimerization, Apt2-C and Apt3-G are closer and hybridize on the cytosine-rich and guanine-rich stretches to trigger FRET between AF488 and Cy3 to indicate the heterodimerization. This probekit possesses Janus-like interactions as Apt2-C can interact with both identical Apt2-C and heterogenous Apt3-G, highly similar to the case that HER2 can both homodimerize identical HER2 and heterodimerize HER3. The probekit’s signals were well matched with HER2:HER2 homodimerization and HER2:HER3 heterodimerization, enabling more accurate monitoring of them on living tumor cells, offering a potential tool for related tumor assessment and medication.

Proximity-Dependent Switchable ATP Aptasensors Utilizing a High-Performance FRET Reporter.

Here, fluorescent molecular rotors are employed to develop a new type of high-performance FRET system with large Stokes shift, high photostability, and pH insensitivity, showing great promise for use in proximity-dependent DNA aptasensors. Two carboxylated benzothiazole-based molecular rotors are synthesized, displaying bright green and red fluorescence once labeled to DNA. In the proximity state, an efficient FRET occurs between the two dyes, comparable to that of the most commonly used Cy3/Cy5 pair. Similar phenomena are also observed if naphthothiazole-based analogues are adopted. Our developed FRET pair is then attached to the two parts of a split ATP aptamer in a dimeric DNA nanoscaffold controlled by a bimolecular i-motif. In this way, a pH-switched proximity-induced fluorescent ATP aptasensor is constructed, with good sensitivity, selectivity, and reconfiguration. Furthermore, by altering the linker length of the switching unit, the proximity effect is investigated systematically, providing new insight into DNA proximity reactions and their roles in some physiological processes.

Aptamer-Braked Multi-hairpin Cascade Circuits for Logic-Controlled Label-Free In Situ Bioimaging.

As a common hairpin-based amplification strategy, catalytic-hairpin assembly (CHA) has been widely used to construct various DNA circuits for biosensing and imaging. However, the hairpin substrates can potentially react without catalysts and result in circuit leakage, which may be quite severe in a CHA reaction consisting of three or four hairpins due to the formation of stable three-/four-way junction product. To circumvent this problem, here we introduce a well-designed ATP aptamer as a DNA brake into a four-hairpin cascade circuit, where the triggering toehold is blocked by the aptamer brake and thus the circuit leakage decreases dramatically. Such an aptamer-braked DNA circuit is then employed to build an AND logic gate in response to multiple external stimuli in acidic cell membrane microenvironments. Induced by a bimolecular i-motif that binds thioflavin T (ThT), the dimerization of a four-way junction in situ assembled on the cell surface is accomplished, enabling the logic-controlled cell membrane imaging in a label-free manner. Our design would be applicable to other hairpin-based amplification strategies and may find more applications in the construction of multiresponsive DNA cascade circuits in complex living systems.

Extracellular Ion-Responsive Logic Sensors Utilizing DNA Dimeric Nanoassemblies on Cell Surface and Application to Boosting AS1411 Internalization.

High levels of extracellular H+ and K+ are unique features of the tumor microenvironment and have shown great promise for use in cancer-targeted drug delivery. Here, we design H+- and/or K+-responsive logic sensors utilizing in situ dimeric framework nucleic acid (FNA) assembly on the cell surface and for the first time apply the logic sensors to boosting cellular internalization of molecular payloads in tumor-mimicking extracellular environments. An anticancer aptamer AS1411 is blocked on branched FNA vertexes where a bimolecular i-motif is tethered as the controlling unit to enable a dimeric DNA nanoassembly in response to extracellular pH change. K+ promotes AS1411 to fold into a G-quadruplex and thereby release from dimeric FNA in which a proximity DNA hybridization-based FRET happens. Furthermore, such an AND-gated nanosensor functions more efficiently for AS1411 internalization than the conventional pathway. This finding shows significant implications for tumor-microenvironment-recognizing target drug delivery and precision cancer therapy.

A bimolecular i-motif mediated FRET strategy for imaging protein homodimerization on a living tumor cell surface.

A bimolecular i-motif mediated FRET strategy was developed based on the proximity-induced folding of two identical cytosine-rich DNA strands. This strategy affords a FRET signal that is highly matched to the dimerization event, and enabled accurate and dynamic in situ imaging of Met homodimerization on a living tumor cell surface.

Exploring potential of i-motif DNA formed in the promoter region of GRIN1 gene for nanotechnological applications

The tandem repeat stretches of cytosine residues fold to construct intercalated motif (i-motif) structures over an acidic pH range. Several advancements have been made to understand the biological role of i-motif DNA. Potential i-motif structures found in gene promoters are involved in positive transcriptional regulation and sequence specific DNA binding. Herein, a 27-mer C-rich DNA stretch present at the promoter region of (Glutamate Ionotropic Receptor NMDA type subunit 1) GRIN1 gene, named GRIN1c was studied to investigate its structural status using biophysical and biochemical techniques. The gel electrophoresis, circular dichroism spectroscopy and pH-dependent monophasic UV-thermal melting data revealed that GRIN1c sequence adapts a bimolecular i-motif structure. Two alternative structural models are suggested for the bimolecular i-motif structure with typical C·C+ base pairing and significant Watson-Crick base pairing. Possibility of co-existence of non-Watson-Crick and Watson-Crick base pairing in the formation of i-motif expands the repertoire of various topologies of i-motif structures. Extensive applications of i-motif structure in the areas of nanotechnology, analytical and biomedical systems and many more have made it essential to explore these multistranded structures. Nanodevices-based on i-motif structure found applications as biosensors and drug release platform, and also serve as proof for their in vivo existence.

Reconfigurable Bioinspired Framework Nucleic Acid Nanoplatform Dynamically Manipulated in Living Cells for Subcellular Imaging.

In nature, the formation of spider silk fibers begins with dimerizing the pH-sensitive N-terminal domains of silk proteins (spidroins) upon lowering pH, and provides a natural masterpiece for programmable assembly. Inspired by the similarity of pH-dependent dimerization behaviors, introduced here is an i-motif-guided model to mimic the initial step of spidroin assembly at the subcellular level. A framework nucleic acid (FNA) nanoplatform is designed using two tetrahedral DNA nanostructures (TDNs) with different branched vertexes carrying a bimolecular i-motif and a split ATP aptamer. Once TDNs enter acidic lysosomes within living cells, they assemble into a heterodimeric architecture, thereby enabling the formation of a larger-size framework and meanwhile subcellular imaging in response to endogenous ATP, which can be dynamically manipulated by adjusting intracellular pH and ATP levels with external drug stimuli.

Structural polymorphism of a cytosine-rich DNA sequence forming i-motif structure: Exploring pH based biosensors

Sequence recognition and conformational polymorphism enable DNA to emerge out as a substantial tool in fabricating the devices within nano-dimensions. These DNA associated nano devices work on the principle of conformational switches, which can be facilitated by many factors like sequence of DNA/RNA strand, change in pH or temperature, enzyme or ligand interactions etc. Thus, controlling these DNA conformational changes to acquire the desired function is significant for evolving DNA hybridization biosensor, used in genetic screening and molecular diagnosis. For exploring this conformational switching ability of cytosine-rich DNA oligonucleotides as a function of pH for their potential usage as biosensors, this study has been designed. A C-rich stretch of DNA sequence (5′-TCCCCCAATTAATTCCCCCA-3′; SG20c) has been investigated using UV-Thermal denaturation, poly-acrylamide gel electrophoresis and CD spectroscopy. The SG20c sequence is shown to adopt various topologies of i-motif structure at low pH. This pH dependent transition of SG20c from unstructured single strand to unimolecular and bimolecular i-motif structures can further be exploited for its utilization as switching on/off pH-based biosensors.

Programmable i-motif DNA folding topology for a pH-switched reversible molecular sensing device.

Four-stranded DNAs including G-quadruplexes and i-motifs are formed from four stretches of identical bases (G or C). A challenge remains in controlling the intermolecular folding of different G-rich or C-rich strands due to the self-association of each component. Here, we introduce a well-designed bimolecular i-motif that does not allow the dimerization of the same strand, and illustrate its usefulness in a pH-switched ATP-sensing DNA molecular device. We analyze two groups of i-motif DNAs containing two stretches of different C-residues (Cn-1TmCn and CnTmCn-1; n = 3−6, m = 1, 3) and show that their bimolecular folding patterns (L- and H-form) noticeably differs in the thermal stability. The L-form structures generally display a relatively low stability, with a bigger difference from that of conventional i-motifs formed by CnTmCn. It inspires us to at utmost improving the structural stability by extending the core of L-form bimolecular i-motifs with a few flanking noncanonical base pairs, and therefore to avoid the dimeric association of each component. This meaningful bimolecular i-motif is then incorporated into a three-way junction (3WJ) and a four-way junction (4WJ) functionalized with two components of a ATP-binding split DNA aptamer, allowing the pH-triggered directional assembly of 3WJ and 4WJ into the desired (3+4)WJ structure that is verified by gel electrophoresis. It therefore enables the ATP-induced association of the split aptamer within the (3+4)WJ structure, as monitored by fluorescence quenching. In this way, the designed DNA system behaves as a pH-switched reversible molecular device, showing a high sensitivity and selectivity for fluorescent ATP analysis. The i-motif folding topology-programmed DNA nanoassembly may find more applications in the context of larger 2D/3D DNA nanostructures like lattices and polyhedra.

Self-assembly and nanoaggregation of a pH responsive DNA hybrid amphiphile.

This work describes the design and preparation of a simple but novel hybrid amphiphile containing a pH-responsive DNA sequence. The formation of a bimolecular i-motif structure allows the control of reversible switching of the hybrid amphiphile between the dimer and unimer by pH. Thus, spherical aggregates with distinct self-assembly pathways, sizes and structures are obtained at pH 4.5 and pH 9.0, and the structures can be switched by the change of pH and thermal annealing. This work reports different self-assembled nanostructures and their transitions that give this amphiphile potential for the design of controllable drug delivery systems.

Interlocked DNA nanostructures controlled by a reversible logic circuit.

DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems.

Structural transition from dimeric to tetrameric i‐motif, caused by the presence of TAA at the 3′‐end of human telomeric C‐rich sequence

Widely dispersed in genomic DNA, the tandem C-rich repetitive stretches may fold below physiological pH, into i-motif structures, stabilized by C.C(+) pairing. Herein, structural status of a 9-mer stretch d(CCCTAACCC), [the truncated double repeat of human telomeric sequence], and its extended version, comprising of additional--TAA segment at the 3'-end, representing the complete double repeat d(CCCTAACCCTAA), has been investigated. The pH dependent monophasic UV-melting, Gel and CD data suggested that while the truncated version adopts a bimolecular i-motif structure, its complete double repeat (12-mer) sequence exists in two (bimolecular and tetramolecular) forms. A model is proposed for the tetramolecular i-motif with conventional C.C(+) base pairs, additionally stabilized by asymmetric A.A base pairs at the -3' TAA flanking ends and Watson-Crick A.T hydrogen bonding between intervening bases on antiparallel strands. Expanding the known topologies of DNA i-motifs, such atypical geometries of i-motifs may have implications in their recognition by proteins.

D-2-Deoxyribose and D-Arabinose, but not D-Ribose, Stabilize the Cytosine Tetrad (i-DNA) Structure

Described here are studies exploring the effect of the sugar-phosphate backbone on the stability of i-tetrads in solution [K. Gehring et al. Nature 363, 561–565 (1993)]. In the accompanying paper, branched oligonucleotides are shown to be effective probes for organizing oligodeoxycytidine strands into I-motif structures (C-tetrads). Specifically, the joining of a pair of parallel deoxycytidylate strands with a riboadenosine “linker” leads to marked enhancement in stability of the tetrad structure. To further characterize the nature of the sugar-sugar interactions in this novel structure, branched oligonucleotides containing D-arabinocytidine and D-ribocytidine were synthesized and their association properties examined. The ribo oligomers were prepared in two regioisomeric forms differing only in the connectivities of the deoxycytidine strands, i.e., 3′-to-5′ versus 2′-to-5′ linked dC5 strands. The branched D-deoxycytidine analogue, rA(2′,5′-dC5)3′,5′-dC5, which previously has been shown to fold into a bimolecular I-motif, served as model system. It is found that the arabinose substitution leads to hypochromic structures that are characteristic of four-stranded intercalated DNA and has little, if any, effect on the stability of the complex formed. Parallel experiments with the branched ribocytidine analogs gave very weak or no discernible UV transitions, consistent with no strand association in this case [Lacroix et al., Biochemistry 35, 8715–8722 (1996)]. These results are discussed in relation to expected steric interactions of oligocytidine strands within the I-structure. The findings increase our understanding of the impact of the sugar and intemucleotide connectivity on the stability of this higher-order nucleic acid structure.