5‐Formyluracil as a Multifunctional Building Block in Biosensor Designs

In organisms 5‐formyluracil (5fU), which is known as a vital natural nucleobase, is widely present. Despite the recent development of sensor designs for organic fluorescent molecules for selective targeting applications, biocompatible and easily operated probe designs that are based on natural nucleobase modifications have rarely been reported. Here, we introduce the idea of 5fU as a multifunctional building block to facilitate the design and synthetic development of biosensors. The azide group was derived from the sugar of a nucleoside, which can be further used in the selective binding of cells or organelles through click chemistry with alkynyl‐modified targeting groups. The aldehyde group of 5fU can react with different chemicals to generate environmentally sensitive nucleobases that have obvious characteristics, which precious reactants cannot achieve for selective fluorogenic switch‐on detection of a specific target. We first synthesized 5fU analogues that had aggregation‐induced emission properties, and then we used triphenylphosphonium as a mitochondria‐targeting group to selectively image mitochondria in cancer cells and mouse embryonic stem cells. Additionally, the reagents exhibit a high selectivity for reaction with 5fU, which means that the method can also be used for the detection of 5fU. Combining the two characteristics, the idea of 5fU as a multifunctional building block in biosensor designs may potentially be applicable in 5fU site‐specific microenvironment detection in future research.     

Qualitative and quantitative detection of aldehydes in DNA with 2-amino benzamidoxime derivative

An aldehyde-reactive probe based on 2-amino benzamidoxime (ABAO) framework was introduced, which can selectively label aldehydes in DNA through intramolecular ring closure under mild aqueous solutions. We screened ABAO derivatives that can undergo a cyclization with the formylated nucleobases to generate a fluorescence nucleoside, and of these derivatives 5?methoxy-ABAO (PMA) emerged as the optimal choice. PMA can sensitively and selectively react with 5fU, 5fC and AP to form fluorogenic dihydroquinazoline derivatives, which also can quantify DNA damages induced by γ-irradiation. PMA-initiated labeling strategy provides great convenience for qualitative and quantitative detection of aldehydes in DNA.     

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.     

A photo-elutable and template-free isothermal amplification strategy for sensitive fluorescence detection of 5-formylcytosine in genomic DNA

5-Formylcytosine (5fC), as an important epigenetic modification, plays a vital role in diverse biological processes and multiple diseases by regulating gene expression. Owing to the extremely low abundance of 5fC in all mammalian tissues and high structural similarity with other cytosine derivatives, the precise and sensitive detection of 5fC is challenging. Herein, a photo-elutable and template-free isothermal amplification strategy has been proposed for the sensitive detection of 5fC in genomic DNA based on 5fC-specific biotinylation, enrichment, photocleavage, and terminal deoxynucleotidyl transferase (TdT)-assisted fluorescence signal amplification, which is termed 5fC-PTIAS. By introducing the highly specific chemolabeling and the one-step photoelution processes, this strategy possesses a minimal nonspecific background as well as a much higher amplification efficiency. With the high signal-to-noise ratio, this strategy can achieve the accurate quantification of 5fC in various biological samples including mouse brain, kidney, and liver, with a limit of detection (LOD) of 0.025‰ in DNA (S/N = 3). These results not only confirm the widespread distribution of 5fC but also indicate its significant variation in different tissues and ages. The bisulfite- and mass spectrometry-free strategy is highly sensitive, selective, and easily mastered, holding great promise in detecting other epigenetic modifications with much lower levels.     

Unnatural Cytosine Bases Recognized as Thymines by DNA Polymerases by the Formation of the Watson–Crick Geometry

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms by which DNA polymerases faithfully decode chemical information on the template. It was previously shown that two unnatural cytosine bases (termed “M‐fC” and “I‐fC”), which are chemical labeling adducts of the epigenetic base 5‐formylcytosine, can induce C‐to‐T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remains enigmatic. Herein, crystal structures of unnatural cytosine bases pairing to dA/dG in the KlenTaq polymerase‐host–guest complex system and pairing to dATP in the KlenTaq polymerase active site were determined. Both M‐fC and I‐fC base pair with dA/dATP, but not with dG, in a Watson–Crick geometry. This study reveals that the formation of the Watson–Crick geometry, which may be enabled by the A‐rule, is important for the recognition of unnatural cytosines.     

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Methyltransferases have proven useful to install functional groups site‐specifically in different classes of biomolecules when analogues of their cosubstrate S‐adenosyl‐l ‐methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site‐specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell‐permeable, thus preventing implementation of this strategy in cells. We present a two‐step enzymatic cascade for site‐specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5′ cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.     

5-Formyluracil targeted biochemical reactions with proteins inhibit DNA replication,induce mutations and interference gene expression in living cells

Covalent DNA–protein cross-links are toxic DNA lesions that interfere with essential biological processes, which can cause serious biological consequences, such as genomic instability and protein misexpression. 5-Formyluracil (5fU) as an important modification in DNA, which is mainly from oxidative damage, exists in a variety of cells and tissues. We have reported that 5fU mediated DNA–protein conjugates could exist in human cells [Zhou et al. CCS Chem. 2 (2020) 54–63]. We now aimed to explore its potential biological effects in vitro and in vivo. In this paper, we firstly reported that 5fU intermediated DNA–peptide or DNA–protein conjugates (both were called DPCs) could inhibit different polymerases bypass or cause mutations. Then we further investigated the functional impacts caused by 5fU-mediated DPCs, which appeared in different gene expression components [in the promoter sequence or 5′-untranslated regions (UTR)]. These results together may contribute to a broader understanding of DNA–protein interactions as well as the biological functions associated with 5fU.     

Butylacrylate‐nucleobase Conjugates as Targets for Two‐step Redox Labeling of DNA with an Osmium Tetroxide Complex

Modification of nucleic acids with osmium tetroxide reagents (Os,L, such as OsO₄,2,2′‐bipyridine, Os,bpy) has been applied in redox DNA labeling, in probing DNA structure as well as in studies of DNA interactions with other molecules. In natural DNA, primarily thymine residues form adducts with the Os,bpy in a structure selective manner. In this paper we introduce a new two‐step technique of DNA modification with the electroactive Os,bpy, consisting in enzymatic construction of DNA bearing butyl acrylate (BA) moieties attached to uracil at C5 or to 7‐deaza adenine at C7, followed by chemical modification of a reactive C=C double bond in the acrylate residue. We demonstrate a facile modification of the BA conjugates in both single‐ and double‐stranded (ds) DNA under conditions when modification within the nucleobase rings in ds DNA is hindered. Various DNA−Os,bpy adducts can easily be analyzed electrochemically and distinguished by different redox potentials. The two‐step procedure appears to be applicable in osmium redox labelling of ds DNA.     

DNAzyme-Catalyzed Site-Specific N-Acylation of DNA Oligonucleotide Nucleobases

We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5′-carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.     

Rational Design for Cooperative Recognition of Specific Nucleobases Using β‐Cyclodextrin‐Modified DNAs and Fluorescent Ligands on DNA and RNA Scaffolds

We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a β‐cyclodextrin (β‐CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase‐specific heterocycle and an environment‐sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby β‐CyD. Three reporter ligands, MNDS (naphthyridine–dansyl linked ligand), MNDB (naphthyridine–DBD), and DPDB (pyridine–DBD), were used for DNA and RNA probing with 3′‐end or 5′‐end modified β‐CyD – ODN conjugates. For the DNA target, the β‐CyD tethered to the 3′‐end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site ( MNDS and DPDB ). Meanwhile the β‐CyD on the 5′‐end of the ODN interacted with the fluorophore in the minor groove ( MNDB and DPDB ). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules.     

Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine

5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.     

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

5‐hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non‐enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein–DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single‐base resolution. We employ chemical oxidation to transform 5hmU to 5‐formyluracil (5fU), followed by the polymerase extension to induce T‐to‐C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T‐to‐C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU‐rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.     

Chemoselective Modification of Vinyl DNA by Triazolinediones

A new method for the post‐synthetic modification of nucleic acids was developed that involves mixing a phenyl triazolinedione (PTAD) derivative with DNA containing a vinyl nucleobase. The resulting reactions proceeded through step‐wise mechanisms, giving either a formal [4+2] cycloaddition product, or, depending on the context of nucleobase, PTAD addition along with solvent trapping to give a secondary alcohol in water. Catalyst‐free addition between PTAD and the terminal alkene of 5‐vinyl‐2′‐deoxyuridine (VdU) was exceptionally fast, with a second‐order rate constant of 2×10³ m ⁻¹ s⁻¹. PTAD derivatives selectively reacted with VdU‐containing oligonucleotides in a conformation‐selective manner, with higher yields observed for G‐quadruplex versus duplex DNA. These results demonstrate a new strategy for copper‐free bioconjugation of DNA that can potentially be used to probe nucleic acid conformations in cells.     

Pyrene‐Based Quantitative Detection of the 5‐Formylcytosine Loci Symmetry in the CpG Duplex Content during TET‐Dependent Demethylation

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten‐eleven‐translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence‐based probe not only to target 5‐formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET‐dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET‐dependent 5mC oxidation and novel mechanistic insights into the TET‐dependent 5mC oxidation in the mCpG context.     

Nucleobase and ribose modifications control immunostimulation by a microRNA-122-mimetic RNA

Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2'-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory 'hot spots' within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation.     

Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site‐specific inhibition and timed removal. S‐Adenosyl‐L‐methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC‐ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC‐ChMAT at 1.87 Å revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC‐MATs were compatible with DNA‐ and RNA‐MTases, enabling sequence‐specific modification (“writing”) of plasmid DNA and light‐triggered removal (“erasing”).     

Reversed Assembly of Dyes in an RNA Duplex Compared with Those in DNA

We prepared reversed dye clusters by hybridizing two RNA oligomers, each of which tethered dyes (Methyl Red, 4′‐methylthioazobenzene, and thiazole orange) on D ‐threoninols (threoninol nucleotides) at the center of their strands. NMR spectroscopic analyses revealed that two dyes from each strand were axially stacked in an antiparallel manner to each other in the duplex, and were located adjacent to the 3′‐side of a natural nucleobase. Interestingly, this positional relationship of the dyes was completely the opposite of that assembled in DNA that we reported previously: dyes in DNA were located adjacent to the 5′‐side of a natural nucleobase. This observation was also consistent with the circular dichroism of dimerized dyes in which the Cotton effect of the dyes (i.e., the winding properties of two dyes) was inverted in RNA relative to that in DNA. Further spectroscopic analyses revealed that clustering of the dyes on RNA duplexes induced distinct hypsochromicity and narrowing of the band, thus demonstrating that the dyes were axially stacked (i.e., H‐aggregates) even on an A‐type helix. On the basis of these results, we also prepared heterodimers of a fluorophore (thiazole orange) and quencher (Methyl Red) in an RNA duplex. Fluorescence from thiazole orange was found to be strongly quenched by Methyl Red due to the excitonic interaction, so that the ratio of fluorescent intensities of the RNA–thiazole orange conjugate with and without its complementary strand carrying a quencher became as high as 27. We believe that these RNA–dye conjugates are potentially useful probes for real‐time monitoring of RNA interference (RNAi) mechanisms.     

Azido‐Functionalized 5′ Cap Analogues for the Preparation of Translationally Active mRNAs Suitable for Fluorescent Labeling in Living Cells

The 7‐methylguanosine (m⁷G) cap structure is a unique feature present at the 5′ ends of messenger RNAs (mRNAs), and it can be subjected to extensive modifications, resulting in alterations to mRNA properties (e.g. translatability, susceptibility to degradation). It also can provide molecular tools to study mRNA metabolism. We developed new mRNA 5′ cap analogues that enable the site‐specific labeling of RNA at the 5′ end using strain‐promoted azide–alkyne cycloaddition (SPAAC) without disrupting the basic function of mRNA in protein biosynthesis. Some of these azide‐functionalized compounds are equipped with additional modifications to augment mRNA properties. The application of these tools was demonstrated by labeling translationally active mRNAs in living cells.