Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Imidazolo‐dC Metal‐Mediated Base Pairs: Purine Nucleosides Capture Two Ag+ Ions and Form a Duplex with the Stability of a Covalent DNA Cross‐Link

8‐Phenylimidazolo‐dC (^phImidC, 2 ) forms metal‐mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent “purine” 2′‐deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6 . Owing to the ease of nucleobase deprotonation, the new Ag⁺‐mediated base pair containing a “purine” skeleton is much stronger than that derived from the pyrrolo‐ [3,4‐d]pyrimidine system (^phPyrdC, 1 ). The silver‐mediated ^phImidC–^phImidC base pair fits well into the DNA double helix and has the stability of a covalent cross‐link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.     

A Peptide Nucleic Acid (PNA) Heteroduplex Probe Containing an Inosine–Cytosine Base Pair Discriminates a Single‐Nucleotide Difference in RNA

Selective discrimination of a single‐nucleotide difference in single‐stranded DNA or RNA remains a challenge with conventional DNA or RNA probes. A peptide nucleic acid (PNA)‐derived probe, in which PNA forms a pseudocomplementary heteroduplex with inosine‐containing DNA or RNA, effectively discriminates a single‐nucleotide difference in a closely related group of sequences of single‐stranded DNA and/or RNA. The pseudocomplementary PNA heteroduplex is easily converted to a fluorescent probe that distinctively detects a member of highly homologous let‐7 microRNAs.     

Centromeric Alpha‐Satellite DNA Adopts Dimeric i‐Motif Structures Capped by AT Hoogsteen Base Pairs

Human centromeric alpha‐satellite DNA is composed of tandem arrays of two types of 171 bp monomers; type A and type B. The differences between these types are concentrated in a 17 bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C‐rich strand of the two main variants of the human alpha‐satellite A box. We show that, under acidic conditions, the C‐rich strands of two A boxes self‐recognize and form a head‐to‐tail dimeric i‐motif stabilized by four intercalated hemi‐protonated C:C⁺ base pairs. Interestingly, the stack of C:C⁺ base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three‐dimensional structure, although the residues involved in the formation of the i‐motif core are different in each case. Together with previous studies showing that the B box (known as the CENP‐B box) also forms dimeric i‐motif structures, our finding of this non‐canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i‐motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere.     

Photocurrent Generation through Charge‐Transfer Processes in Noncovalent Perylenediimide/DNA Complexes

The charge‐transfer process in noncovalent perylenediimide (PDI)/DNA complexes has been investigated by using nanosecond laser flash photolysis (LFP) and photocurrent measurements. The PDI/DNA complexes were prepared by inclusion of cationic PDI molecules into the artificial cavities created inside DNA. The LFP experiments showed that placement of the PDI chromophore at a specific site and included within the base stack of DNA led to the efficient generation of a charge‐separated state with a long lifetime by photoexcitation. When two PDI chromophores were separately placed at different positions in DNA, the yield of the charge‐separated state with a long lifetime was dependent upon the number of A–T base pairs between the PDIs, which was explained by electron hopping from one PDI to another. Photocurrent generation of the DNA‐modified electrodes with the complex was also dependent upon the arrangement of the PDI chromophores. A good correlation was obtained between observed charge separation and photocurrent generation on the PDI/DNA‐modified electrodes, which demonstrated the importance of the defined arrangement and assembly of organic chromophores in DNA for efficient charge separation and transfer in multichromophore arrays.     

Sequence‐Dependent Duplex Stabilization upon Formation of a Metal‐Mediated Base Pair

An artificial nucleoside surrogate with 1H‐imidazo[4,5‐f][1,10]phenanthroline ( P ) acting as an aglycone has been introduced into DNA oligonucleotide duplexes. This nucleoside surrogate can act as a bidentate ligand, and so is useful in the context of metal‐mediated base pairs. Several duplexes involving a hetero base pair with an imidazole nucleoside have been investigated. The stability of DNA duplexes incorporating the respective Ag^I‐mediated base pairs strongly depends on the sequence context. Quantum mechanical/molecular mechanical (QM/MM) calculations have been performed in order to gain insight into the factors determining this sequence dependence. The results indicated that, in addition to the stabilizing effect that results from the formation of coordinative bonds, destabilizing effects may occur when the artificial base pair does not fit optimally into the surrounding B‐DNA duplex.     

Label‐Free Detection of MicroRNA: Two‐Step Signal Enhancement with a Hairpin‐Probe‐Based Graphene Fluorescence Switch and Isothermal Amplification

A label‐free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA‐21 (mir‐21) as the target to examine the desirable properties of this assay. When the target, as a “trigger”, was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the “signal” amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high‐affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir‐21 at attomole levels. Proof‐of‐concept evidence has been provided for the proposed cost‐effective strategy; thus, this strategy could expand the application of GO‐material‐based bioanalysis for nucleic acid studies.     

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller‐shaped tetraphenylethene (TPE) salts with aggregation‐induced emission (AIE) characteristics. 1,1,2,2‐Tetrakis[4‐(2‐triethylammonioethoxy)phenyl]ethene tetrabromide (TPE‐ 1 ) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady‐state and time‐resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE‐ 1 enables it to serve as a bioprobe for direct monitoring of cation‐driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G‐quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE‐ 1 is cytocompatible, posing no toxicity to living cells.     

Stabilization of G‐Quadruplex DNA with Platinum(II) Schiff Base Complexes: Luminescent Probe and Down‐Regulation of c‐myc Oncogene Expression

The interactions of a series of platinum(II) Schiff base complexes with c‐myc G‐quadruplex DNA were studied. Complex [PtL ^1a ] ( 1 a ; H₂L ^1a =N,N′‐bis(salicylidene)‐4,5‐methoxy‐1,2‐phenylenediamine) can moderately inhibit c‐myc gene promoter activity in a cell‐free system through stabilizing the G‐quadruplex structure and can inhibit c‐myc oncogene expression in cultured cells. The interaction between 1 a and G‐quadruplex DNA has been examined by ¹H NMR spectroscopy. By using computer‐aided structure‐based drug design for hit‐to‐lead optimization, an in silico G‐quadruplex DNA model has been constructed for docking‐based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL ³ ] ( 3 ; H₂L ³ = N,N′‐bis{4‐[1‐(2‐propylpiperidine)oxy]salicylidene}‐4,5‐methoxy‐1,2‐phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c‐myc G‐quadruplex. The inhibitory activity of 3 (IC₅₀=4.4 μM ) is tenfold more than that of 1 a . The interaction between 1 a or 3 with c‐myc G‐quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1 a and 3 bind c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3’ terminal face of the G‐quadruplex. Such binding of G‐quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at λ_max=652 nm. Complex 3 also effectively down‐regulated the expression of c‐myc in human hepatocarcinoma cells.     

Construction of a Near‐Infrared Fluorescent Turn‐On Probe for Selenol and Its Bioimaging Application in Living Animals

As selenocysteine (Sec) carries out the majority of the functions of the various Se‐containing species in vivo, it is of high importance to develop reliable and rapid assays with biocompatibility to detect Sec. Herein, an NIR fluorescent turn‐on probe for highly selective detection of selenol was designed and synthesized. The probe exhibits large turn‐on signal upon treatment with selenocysteine (R‐SeH), and it was further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals.     

Molecular Switching Behavior in Isosteric DNA Base Pairs

The structures and proton‐coupled behavior of adenine–thymine (A‐T) and a modified base pair containing a thymine isostere, adenine–difluorotoluene (A‐F), are studied in different solvents by dispersion‐corrected density functional theory. The stability of the canonical Watson–Crick base pair and the mismatched pair in various solvents with low and high dielectric constants is analyzed. It is demonstrated that A‐F base pairing is favored in solvents with low dielectric constant. The stabilization and conformational changes induced by protonation are also analyzed for the natural as well as the mismatched base pair. DNA sequences capable of changing their sequence conformation on protonation are used in the construction of pH‐based molecular switches. An acidic medium has a profound influence in stabilizing the isostere base pair. Such a large gain in stability on protonation leads to an interesting pH‐controlled molecular switch, which can be incorporated in a natural DNA tract.