Enzymatic Synthesis,Amplification, and Application of DNA with a Functionalized Backbone

The ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4‐3, which better tolerates 2′‐modified substrates. To explore the utility of SFM4‐3, we now report the characterization of its recognition of substrates with 2′‐azido, 2′‐chloro, 2′‐amino, or arabinose sugars. We find that SFM4‐3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4‐3 can also PCR amplify these modified oligonucleotides. When combined with post‐amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel.     

Induction of a Four‐Way Junction Structure in the DNA Palindromic Hexanucleotide 5′‐d(CGTACG)‐3′ by a Mononuclear Platinum Complex

Four‐way junctions (4WJs) are supramolecular DNA assemblies comprising four interacting DNA strands that in biology are involved in DNA‐damage repair. In this study, a new mononuclear platinum(II) complex 1 was prepared that is capable of driving the crystallization of the DNA oligomer 5′‐d(CGTACG)‐3′ specifically into a 4WJ‐like motif. In the crystal structure of the 1 –CGTACG adduct, the distorted‐square‐planar platinum complex binds to the core of the 4WJ‐like motif through π–π stacking and hydrogen bonding, without forming any platinum–nitrogen coordination bonds. Our observations suggest that the specific molecular properties of the metal complex are crucially responsible for triggering the selective assembly of this peculiar DNA superstructure.     

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.     

A Biocompatible Heterogeneous MOF–Cu Catalyst for In Vivo Drug Synthesis in Targeted Subcellular Organelles

As a typical bioorthogonal reaction, the copper‐catalyzed azide–alkyne cycloaddition (CuAAC) has been used for drug design and synthesis. However, for localized drug synthesis, it is important to be able to determine where the CuAAC reaction occurs in living cells. In this study, we constructed a heterogeneous copper catalyst on a metal–organic framework that could preferentially accumulate in the mitochondria of living cells. Our system enabled the localized synthesis of drugs through a site‐specific CuAAC reaction in mitochondria with good biocompatibility. Importantly, the subcellular catalytic process for localized drug synthesis avoided the problems of the delivery and distribution of toxic molecules. In vivo tumor therapy experiments indicated that the localized synthesis of resveratrol‐derived drugs led to greater antitumor efficacy and minimized side effects usually associated with drug delivery and distribution.     

Steering Siglec–Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry

Sialic acid sugars that terminate cell‐surface glycans form the ligands for the sialic acid binding immunoglobulin‐like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross‐reactivity and led to the discovery of three selective Siglec‐5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec‐3 dampened the activation of Siglec‐3⁺ monocytic cells through the NF‐κB and IRF pathways.     

Copper–Alkyne Complexation Responsible for the Nucleolar Localization of Quadruplex Nucleic Acid Drugs Labeled by Click Reactions

G‐Quadruplex(es) (G4) are noncanonical nucleic‐acid structures found in guanine‐rich sequences. They can be targeted with small molecules (G4 ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localization of PhenDC₃, one of the most powerful G4 ligands, by synthesizing two clickable azide and alkyne derivatives (PhenDC₃‐alk, PhenDC₃‐az) and labeling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper‐based CuAAC and copper‐free SPAAC methodologies in fixed cells implicated Cu^I/alkyne intermediates in the nonspecific localization of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labeling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localize drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.     

Imaging Endogenous Metal Ions in Living Cells Using a DNAzyme–Catalytic Hairpin Assembly Probe

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme‐based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na⁺‐specific DNAzyme to detect endogenous Na⁺ inside cells is reported. Upon light activation and in the presence of Na⁺, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na⁺ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.     

A Simple,High‐Yield Synthesis of DNA Duplexes Containing a Covalent,Thermally Cleavable Interstrand Cross‐Link at a Defined Location

Interstrand DNA–DNA cross‐links are highly toxic to cells because these lesions block the extraction of information from the genetic material. The pathways by which cells repair cross‐links are important, but not well understood. The preparation of chemically well‐defined cross‐linked DNA substrates represents a significant challenge in the study of cross‐link repair. Here a simple method is reported that employs “post‐synthetic” modifications of commercially available 2′‐deoxyoligonucleotides to install a single cross‐link in high yield at a specified location within a DNA duplex. The cross‐linking process exploits the formation of a hydrazone between a non‐natural N⁴‐amino‐2′‐deoxycytidine nucleobase and the aldehyde residue of an abasic site in duplex DNA. The resulting cross‐link is stable under physiological conditions, but can be readily dissociated and re‐formed through heating–cooling cycles.