High‐Density DNA Functionalization by a Combination of Cu‐Catalyzed and Cu‐Free Click Chemistry

We report the regioselective Cu‐free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3‐Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5‐disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu‐free and Cu‐catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high‐density functionalization of DNA is simple, modular, and efficient.     

An Isotope‐Coded Fluorogenic Cross‐Linker for High‐Performance Target Identification Based on Photoaffinity Labeling

A photoaffinity labeling (PAL)‐based method for the rapid identification of target proteins is presented in which a high‐performance chemical tag, an isotope‐coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target‐selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins.     

Affinity‐Labeling‐Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site‐specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity‐labeling‐based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime‐exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target‐specific multiple modification in a protein mixture. The availability of various (photo)affinity‐labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site‐selective incorporation of many functional molecules into proteins.     

A Light‐Up Probe with Aggregation‐Induced Emission for Real‐Time Bio‐orthogonal Tumor Labeling and Image‐Guided Photodynamic Therapy

Bio‐orthogonal tumor labeling is more effective in delivering imaging agents or drugs to a tumor site than active targeting strategy owing to covalent ligation. However, to date, tumor‐specific imaging through bio‐orthogonal labeling largely relies on body clearance to differentiate target from the intrinsic probe signal owing to the lack of light‐up probes for in vivo bio‐orthogonal labeling. Now the first light‐up probe based on a fluorogen with aggregation‐induced emission for in vivo bio‐orthogonal fluorescence turn‐on tumor labeling is presented. The probe has low background fluorescence in aqueous media, showing negligible non‐specific interaction with normal tissues. Once it reacts with azide groups introduced to tumor cells through metabolic engineering, the probe fluorescence is lightened up very quickly, enabling rapid tumor‐specific imaging. The photosensitizing ability was also used to realize effective image‐guided photodynamic tumor therapy.     

Synthesis of N,N‐Dialkylamino‐nor‐Dihydroxanthene‐Hemicyanine Fused Near‐Infrared Fluorophores and Their First Water‐Soluble and/or Bioconjugatable Analogues

The effective synthesis of extended conjugated N ,N ‐dialkylamino‐nor ‐dihydroxanthene‐based fluorophores is described from diversely functionalized salicylic aldehydes. The access to these original fluorescent derivatives proceeded in two steps through a one‐pot construction of the unusual nor ‐dihydroxanthene (nor ‐DHX) scaffold followed by a diversification step providing a wide variety of nor ‐DHX‐hemicyanine fused dyes emitting in the range of 730–790 nm. The versatility of our approach has enabled a further extension to the late‐stage introduction of negatively/positively charged polar groups onto their terminal nitrogen heterocyclic subunit, thereby giving access to the first water‐soluble and/or bioconjugatable members of this emerging class of NIR fluorophores. Our water‐solubilizing method is easily implementable, and the nor ‐DHX‐hemicyanine skeleton maintains satisfying fluorescence quantum yields (5–20 %) under physiological conditions. Finally, the bioconjugation ability of fluorescent derivatives bearing a free carboxylic acid was demonstrated through the covalent labeling of a model protein, namely, bovine serum albumin.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

One‐Step Formation of “Chain‐Armor”‐Stabilized DNA Nanostructures

DNA‐based self‐assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA‐based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6‐helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3′‐ends and azides on their 5′‐ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so‐called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.     

Universal pH‐Responsive and Metal‐Ion‐Free Self‐Assembly of DNA Nanostructures

pH‐responsiveness has been widely pursued in dynamic DNA nanotechnology, owing to its potential in biosensing, controlled release, and nanomachinery. pH‐triggering systems mostly depend on specific designs of DNA sequences. However, sequence‐independent regulation could provide a more general tool to achieve pH‐responsive DNA assembly, which has yet to be developed. Herein, we propose a mechanism for dynamic DNA assembly by utilizing ethylenediamine (EN) as a reversibly chargeable (via protonation) molecule to overcome electrostatic repulsions. This strategy provides a universal pH‐responsivity for DNA assembly since the regulation originates from externally co‐existing EN rather than specific DNA sequences. Furthermore, it endows structural DNA nanotechnology with the benefits of a metal‐ion‐free environment including nuclease resistance. The concept could in principle be expanded to other organic molecules which may bring unique controls to dynamic DNA assembly.     

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.     

Artificial Cysteine S‐Glycosylation Induced by Per‐O‐Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling

The unexpected, non‐enzymatic S‐glycosylation of cysteine residues in various proteins by per‐O‐acetylated monosaccharides is described. This artificial S‐glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per‐O‐acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O‐GlcNAcylated proteins and O‐GlcNAc sites in HeLa cells has been assembled by using N‐azidoacetylgalactosamine (GalNAz).     

Tag‐Convertible Photocrosslinker with Click‐On/Off N‐Acylsulfonamide Linkage for Protein Identification

The N‐acylsulfonamide group, known as a safety‐catch linker, has been applied to photoaffinity labeling (PAL) using a cinnamate‐type photocrosslinker to improve the efficiency of PAL‐based target identification. A bioorthogonal sulfo‐click reaction was used to stably link a photocrosslinker unit with N‐acylsulfonamide linkage to produce a photoactivatable probe without any protection. In addition, the crosslinked protein was selectively isolated with a small cinnamate tag via linkage disruption upon N‐alkylation. Furthermore, the tag moiety was photochemically converted to a stable coumarin derivative by losing a water molecule, which is a useful property in MS‐based identification.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.