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.

DNA logic nanodevices that in situ operate within living cells have attracted increasing interest and shown great promise for gene regulation and target recognition. A challenge remains how to control their activation inside specific cellular compartments. Toward this goal, here we report a lysosomerecognizing framework nucleic acid (FNA) nanodevice using an i-motif and an ATP-binding aptamer (ABA) incorporated into a DNA triangular prism (DTP) as the logic-controlling units. Once entering the lysosomal compartments, the FNA device responds to lysosomal pH and ATP via the folding of i-motif and ABA, which triggers a structural change of FNA and the release of a reporter structure for subcellular imaging. With endogenous proton and ATP as two inputs, an AND logic gate is built and in situ operated within living lysosomes by pH and ATP modulation with external drug stimuli. Given the abnormal levels of pH and ATP within some cancer cells or dysfunctional lysosomal cells, in this context our designed FNA logic device may find extended applications in controllable drug release and disease treatment.

Programming intelligent DNA nanocarriers for the targeted transport of molecular payloads in living cells has attracted extensive attention. In vivo activation of these nanocarriers usually relies on external light irradiation. An interest is emerging in the automatic recognition of intracellular surroundings by nanocarriers and their in situ activation under the control of programmed DNA‐computation circuits. Herein, we report the integration of DNA circuits with framework nucleic acid (FNA) nanocarriers that consist of a truncated square pyramid (TSP) cage and a built‐in duplex cargo containing an antisense strand of the target mRNA. An i‐motif and ATP aptamer embedded in the TSP are employed as logic‐controlling units to respond to H+ and ATP inside cellular compartments, triggering the release of the sensing element for fluorescent mRNA imaging. Logic‐controlled FNA devices could be used to target drug delivery, enabling precise disease treatment.

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.

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.