RNA is more UV resistant than DNA: the formation of UV-induced DNA lesions is strongly sequence and conformation dependent

DNA and RNA hairpins, which represent well-folded oligonucleotide structures, were irradiated and the amount of damaged hairpins was directly quantified by using ion-exchange HPLC. The types of photoproducts formed in the hairpins were determined by ESI-HPLC-MS/MS experiments. Irradiation of hairpins with systematically varied sequences and conformations (A versus B) revealed remarkable differences regarding the amount of photolesions formed. UV-damage formation is, therefore, a strongly sequence and conformation dependent process.     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.     

Characterization of bleomycin cleavage sites in strongly bound hairpin DNAs

The first complete, systematic study of DNA degradation by bleomycin under conditions analogous to those likely in a therapeutic setting has been carried out. Hairpin DNAs selected for their ability to bind strongly to BLM A(5) were used to determine the relationship between high-affinity DNA binding sites and the cleavage efficiency and selectivity of BLM A(5) and deglycoBLM A(5) on these DNAs. Of the 10 hairpin DNAs examined, 8 contained at least one 5'-GC-3' or 5'-GT-3' cleavage site, which have traditionally been associated with strong cleavage by Fe·BLM. In the hairpin DNAs, these included the strongest cleavage sites for BLM A(5) and were generally among those for deglycoBLM A(5). However, numerous additional cleavages were noted, many at sequences not usually associated with (deglyco)BLM-mediated cleavage. The remaining DNAs lacked the preferred (5'-GC-3' or 5'-GT-3') BLM cleavage sequences; however, strong cleavage was nonetheless observed at a number of unusual cleavage sites. The most prominent cleavage sequences were 5'-AT-3', 5'-AA-3', 5'-GA-3', and 5'-TT-3'; treatment with Fe(II)·BLM A(5) or Fe(II)·deglycoBLM A(5) resulted in strong cleavage at these sequences. Additionally, in contrast with BLM A(5), which produced cleavage within the randomized and flanking invariant regions, deglycoBLM A(5) showed a preference for cleavage in the randomized region of the DNAs. Previous reports have established that deglycoBLM exhibits decreased DNA cleavage efficiency relative to BLM. This was also generally observed when comparing cleavage efficiencies for the strongly bound hairpin DNAs. However, some cleavage bands produced by Fe·deglycoBLM A(5) were stronger in intensity than those produced by BLM A(5) at concentrations optimal for both compounds. To investigate the chemistry of DNA degradation, selected hairpin DNAs were treated with n-butylamine following cleavage with Fe(II)·BLM A(5) or Fe(II)·deglycoBLM A(5) to explore the alkali labile pathway of DNA degradation by BLM. While all 10 DNAs showed evidence of alkali labile products, five DNA hairpins afforded some products formed solely via the alkali labile pathway.     

Targeting Non‐B‐Form DNA in Living Cells

Under certain conditions, repetitive DNA motifs have the potential to adopt non‐B‐form DNA structures, such as hairpins, triplexes, Z‐DNA, quadruplexes, and i‐motifs. Some non‐B‐form DNAs have been proposed to cause mutations and, consequently, participate in several biologically important processes, including regulation, evolution, and human disease. Advancement in the knowledge of specific interactions between molecules and non‐B‐form DNAs at the molecular level in living cells is important for understanding their biological functions. In this review, we describe the latest studies on molecules that target non‐B‐form DNAs in vivo, with a focus on Z‐DNA, G‐quadruplexes, triplexes, i‐motifs, and hairpins.     

Metallation-Induced Heterogeneous Dynamics of DNA Revealed by Single-Molecule FRET

The metallation of nucleic acids is key to wide-ranging applications, from anticancer medicine to nanomaterials, yet there is a lack of understanding of the molecular-level effects of metallation. Here, we apply single-molecule fluorescence methods to study the reaction of an organo-osmium anticancer complex and DNA. Individual metallated DNA hairpins are characterised using Förster resonance energy transfer (FRET). Although ensemble measurements suggest a simple two-state system, single-molecule experiments reveal an underlying heterogeneity in the oligonucleotide dynamics, attributable to different degrees of metallation of the GC-rich hairpin stem. Metallated hairpins display fast two-state transitions with a two-fold increase in the opening rate to ≈2 s⁻¹, relative to the unmodified hairpin, and relatively static conformations with long-lived open (and closed) states of 5 to ≥50 s. These studies show that a single-molecule approach can provide new insight into metallation-induced changes in DNA structure and dynamics.     

Mismatches Improve the Performance of Strand‐Displacement Nucleic Acid Circuits

Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can potentially react non‐specifically even in the absence of a single‐stranded catalyst, and this non‐specific background degrades the signal‐to‐noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100‐fold increased signal‐to‐background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non‐enzymatic nucleic acid circuits.     

Target‐stimulated DNAzyme Concatamers Released from Aptasensor for Highly Sensitive and Specific Detection of Progesterone

Aiming at the detection of ultralow concentration target progesterone (Pro), a novel electrochemical aptasensor based on DNAzyme concatamers signal amplification strategy was proposed. The strategy consists of target DNA strands (TDNAs), and two different hairpin DNA molecules (H1 and H2). The signal is amplified by the large amount of DNAzyme. The TDNAs modified on the electrode open H1 structures in sequence and propagate a reaction of hybridization events between two alternating hairpins (H1and H2) to obtain abundant DNAzyme concatamers. Upon target Pro introduction, a specific Pro‐TDNAs reaction was executed, thereby resulting in the release of DNAzyme concatamers from the electrode. Subsequent differential pulse voltammetry(DPV) detection of aminoazobenzene (DAP) resulting by DNAzyme catalyze the oxidation of o‐phenylenediamine (OPD) with the aid of hydrogen peroxide (H₂O₂). Likewise, a small amount of target Pro can efficiently induce the release of a large number of the DNAzyme from the electrode in the form of DNAzyme concatamer. Under optimal conditions, the the proposed assay presents good electrochemical responses for determination of target Pro in the range of 0.5 to 15 ng/mL with the detection limit of 0.36 ng/mL. In addition, the resulting sensor can successfully distinguish Pro from coexisting interfering substance and show good stability and high repeatability. What's more, the methodology has also been demonstrated by assaying Pro‐spiked samples in serum.     

A label-free colorimetric platform for DNA via target-catalyzed hairpin assembly and the peroxidase-like catalytic of graphene/Au-NPs hybrids

A target-catalyzed hairpin assembly (CHA) and graphene/Au-NPs hybrids-based platform has been developed for the determination of DNA. This new sensor not only avoided any labeling but also reduced the background signal. In the absence of target, the assembly of H1 and H2 couldn't be triggered. The catalytic activity of graphene/Au-NPs hybrids was inhibited by adsorption of H1 and H2, leading to the “inactive” hybrids unable to catalyze the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB). However, with the addition of target DNA, the target-catalyzed hairpin assembly was initiated and produced plenty of H1–H2 duplex, which had a weak binding affinity with the graphene/Au-NPs. Thus, the protected interface of graphene/Au-NPs hybrids became active and catalyzed the oxidation reaction of TMB accompanied with a colorless to-blue color change. This approach exhibited good sensitivity and specificity for target DNA with a detection limit of 5.74 × 10⁻¹¹ M, and realized the assay of target DNA in human serum samples. Besides, this sensor could be further expanded to detect viruses or proteins by adapting the corresponding aptamers, showing great potential in biochemical detections.     

Catalytic Hairpin Assembly‐Programmed DNA Three‐Way Junction for Enzyme‐Free and Amplified Electrochemical Detection of Target DNA

DNA three‐way junctions (DNA 3WJ) have been widely used as important building blocks for the construction of DNA architectures and dynamic assemblies. Herein, we describe for the first time a catalytic hairpin assembly‐programmed DNA three‐way junction (CHA‐3WJ) strategy for the enzyme‐free and amplified electrochemical detection of target DNA. It takes full advantage of the target‐catalyzed hairpin assembly‐induced proximity effect of toehold and branch‐migration domains for the ingenious execution of the strand displacement reaction to form the DNA 3WJ on the electrode surface. A low detection limit of 0.5 pM with an excellent selectivity was achieved for target DNA detection. The developed CHA‐3WJ strategy also offers distinct advantages of simplicity in probe design and biosensor fabrication, as well as enzyme‐free operation. Thus, it opens a promising avenue for applications in bioanalysis, design of DNA‐responsive devices, and dynamic DNA assemblies.     

Molecular Engineering of DNA: Molecular Beacons

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single‐nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal‐transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem–loop structure holds the fluorescence‐donor and fluorescence‐acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein–DNA interaction studies, and protein recognition.     

Investigation of the pathways of excess electron transfer in DNA with flavin-donor and oxetane-acceptor modified DNA hairpins

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation. Herein we report synthesis of DNA hairpin model compounds containing a flavin as the hairpin head and two different oxetanes in the stem structure of the hairpin. The data show that the electron moves through the duplex even over distances of 17 A. Attempts to trap the moving electron with N2O showed no reduction of the cleavage efficiency showing that the electron moves through the duplex and not through solution. The electron transfer is sequence dependent. The efficiency is reduced by a factor of 2 in GC rich DNA hairpins.     

Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application.     

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

Calorimetric Studies on the pH Dependence in Hairpin Formation within the Human Enkephalin Enhancer Region

Two complementary 23 base-pair oligomers with sequences analogous to the 3, 5-cyclic adenosine monophosphate (cAMP) inducible enhancer region of the human enkaphalin gene were shown previously⁽⁶⁾ to undergo reversible conformational transformation from duplex to two individual hairpin structures, with two GT and two AC base mismatches, respectively. The present study uses differential scanning calorimetry (DSC) to determine H values for both the dissociation of the duplex formed by the complementary stands and the melting of each hairpin structure. Melting of the hairpins was studied at several different pH values and both the H and cooperative unit (CU) parameters for the hairpin with the AC base mismatches was found to be pH dependent. Increased values for H of melting and cooperative unit size at lower pH values supports the possibility of protonation of adenosine bases and formation of stable AC base pairs in this latter hairpin.     

Periodically Ordered,Nuclease‐Resistant DNA Nanowires Decorated with Cell‐Specific Aptamers as Selective Theranostic Agents

DNA nanostructures have shown potential in cancer therapy. However, their clinical application is hampered by the difficulty to deliver them into cancer cells and susceptibility to nuclease degradation. To overcome these limitations, we report herein a periodically ordered nick‐hidden DNA nanowire (NW) with high serum stability and active targeting functionality. The inner core is made of multiple connected DNA double helices, and the outer shell is composed of regularly arranged standing‐up hairpin aptamers. All termini of the components are hidden from nuclease attack, whereas the target‐binding sites are exposed to allow delivery to the cancer target. The DNA NW remained intact during incubation for 24 h in serum solution. Animal imaging and cell apoptosis showed that NWs loaded with an anticancer drug displayed long blood‐circulation time and high specificity in inducing cancer‐cell apoptosis, thus validating this approach for the targeted imaging and therapy of cancers.     

A Molecular Tuning Fork in Single‐Molecule Mechanochemical Sensing

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real‐time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single‐molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence). Reminiscent of a tuning fork that vibrates at a fixed frequency, the device was classified as a molecular tuning fork (MTF). By monitoring the lifetime of the folded and unfolded hairpins with equal populations, we were able to differentiate between the mono‐ and bivalent binding modes during individual antibody‐antigen binding events. We anticipate these mechanospectroscopic concepts and methods will be instrumental for the development of novel bioanalyses.     

Spectroscopic and structural impact of a stem base-pair change in DNA hairpins: GTTC-ACA-GAAC versus GTAC-ACA-GTAC

Successive investigations over the last decade have revealed and confirmed a stable loop closure in a family of d-[GTAC-5Pur6N7N-GTAC] hairpins, where 5Pur6N7N is a AAA, GAG and AXC loop (X being any nucleotide). The trinucleotide loop is characterized by a well defined 5Pur-7N mispairing mode, and by upfield chemical shifts for three sugar protons of the apical nucleotide 6N. The GTTC-ACA-GAAC DNA hairpin, of interest for its likely involvement in Vibrio cholerae genome mutations, has now been investigated. The GTAC-ACA-GTAC DNA hairpin has also been studied because it is intermediate between the other structures, as it contains the loop of the hairpin under consideration and the stem of the above family. The two hairpins with the ACA loop are stable. They show the same mispairing mode and similar upfield shifts as the previous family, but GTTC-ACA-GAAC seems to be slightly less compact than any other. GTTC-ACA-GAAC is remarkable in that it exhibits a B(II) character for the phosphate-ester conformation at 8Gp9A, together with a swing of the upper hairpin into the major groove that, in particular, brings 6CH1' roughly as close to 7AH2 as to 6CH6. These unexpected structural features are qualitatively deduced from (1)H and (31)P NMR spectra, and confirmed by Raman spectroscopy. This comparative study shows that not only the loop sequence but also the stem sequence may control hairpin structures.     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.     

A Near-Infrared Photo-Switched MicroRNA Amplifier for Precise Photodynamic Therapy of Early-Stage Cancers

Stimuli-responsive photodynamic therapy (PDT) is a hot topic in precise medicine, but the low abundance of responsive trigger molecules in early-stage disease limits application. Here we designed an amplifier with multiple upconversion luminances to achieve a near-infrared photo-switched cascade reaction triggered by specific microRNA and precise PDT of early-stage cancers. This amplifier was composed of photo-caged DNA nanocombs and an upconversion nanoparticle (UCNP) sensitized with IRDye 800CW. The nanocomb was prepared by assembling a photozipper-protected hairpin and two kinds of hybridizable hairpin probes on a DNA skeleton. Upon 808-nm light irradiation, the produced UV light cleaved off the photozipper to induce microRNA-responsive cascade hybridization reaction, activating the photosensitizers linked to different hairpins to generate reactive oxygen species (ROS) under the simultaneously emitted blue light for efficient PDT.