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.     

Application of a Highly Robust and Efficient Fluorescence‐Resonance‐Energy‐Transfer (FRET) System in DNA

We report a feasibility study for the application of our newly developed highly efficient and robust fluorescence‐resonance‐energy‐transfer (FRET) system to DNA. A 2′‐oligodeoxynucleotide, 12 , equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions (Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13 ), a significant decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long decay times of the fluorescence of the Ru‐complex, the system allows for highly sensitive time‐gated measurements.     

Single-Molecule FRET Study of DNA G-Quadruplex

ＩｎｔｒｏｄｕｃｔｉｏｎＴｅｌｏｍｅｒｅｓａｒｅｔｈｅｅｎｄｒｅｇｉｏｎｓｏｆｃｈｒｏｍｏｓｏｍｅｓｃｏｎｓｉｓｔｉｎｇｏｆＤＮＡａｎｄａｓｓｏｃｉａｔｅｄｐｒｏｔｅｉｎ .Ｔｈｅｔｅｌｏｍ ｅｒｉｃＤＮＡｃｏｎｔａｉｎｓＧ ｒｉｃｈｒｅｐｅａｔｓｏｆＤＮＡｓｅｑｕｅｎｃｅｓ .ＴｈｉｓＧ ｒｉｃｈｏｖｅｒｈａｎｇｃａｎｆｏｒｍａｓｔａｂｌｅ ｇｕａｎｉｎｅ ｑｕａｄｒｕｐｌｅｘｉｎｖｉｔｒｏｕｎｄｅｒ ｐｈｙｓｉｏｌｏｇｉｃａｌｃｏｎｄｉ ｔｉｏｎｓ[1,2 ] .Ｉｔｉｓｎｏｗｗｅｌｌ ｅｓｔａｂｌｉｓｈｅｄｔ…     

3dDNA: A Computational Method of Building DNA 3D Structures

Considerable progress has been made in the prediction methods of 3D structures of RNAs. In contrast, no such methods are available for DNAs. The determination of 3D structures of the latter is also increasingly needed for understanding their functions and designing new DNA molecules. Since the number of experimental structures of DNA is limited at present, here, we propose a computational and template-based method, 3dDNA, which combines DNA and RNA template libraries to predict DNA 3D structures. It was benchmarked on three test sets with different numbers of chains, and the results show that 3dDNA can predict DNA 3D structures with a mean RMSD of about 2.36 Å for those with one or two chains and fewer than 4 Å with three or more chains.     

Reversible Reconfiguration of DNA Origami Nanochambers Monitored by Single‐Molecule FRET

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance‐dependent energy transfer between two preloaded fluorophores. Interestingly, single‐molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine‐tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.     

Fluorescence resonance energy transfer in dye-labeled DNA

We present the results of molecular modeling of dye-labeled, double-stranded DNA. The structural information obtained from the simulations are used as input to an analysis of energy transfer in this system. The simulations reveal the nature of the interaction between a pair of fluorophores and DNA. The donor, tetramethylrhodamine, TMR, attached to the 5′-end of DNA with a six-carbon tether, interacts primarily with DNA's minor groove, but occasionally stacks against the DNA base pairs. The acceptor, Cy5, attached to the opposite strand at positions n (n = 7, 12, 14, 16, 19, 24, 27), binds in the major groove in two distinct locations on the upper and lower part of the groove. We analyzed in detail the dye-to-dye distances, dipole orientation factors and fluorescence resonance energy transfer (FRET) rates. Tests of the validity of the Förster model were conducted using the transition density cube (TDC) method, which provides the exact Coulombic interaction within a certain model chemistry. Our studies show that the use of long tethers does not guarantee rotational freedom of the dyes, as intended in the experiments. Instead, the tethers allow Cy5 to bind in two different geometries, which causes a large uncertainty in the dye-to-dye distances. Our results also show significant fluctuation in the orientation factor, κ², which, together with uncertainty in dye-to-dye distances, cause considerable uncertainty in interpreting FRET measurements. We suggest that molecular modeling, combined with the TDC method, provides a useful tool in designing and interpreting FRET experiments.     

Metal-induced specific and nonspecific oligonucleotide folding studied by FRET and related biophysical and bioanalytical implications

Metal induced nucleic acid folding has been extensively studied with ribozymes, DNAzymes, tRNA and riboswitches. These RNA/DNA molecules usually have a high content of double-stranded regions to support a rigid scaffold. On the other hand, such rigid structural features are not available for many in vitro selected or rationally designed DNA aptamers; they adopt flexible random coil structures in the absence of target molecules. Upon target binding, these aptamers adaptively fold into a compact structure with a reduced end-to-end distance, making fluorescence resonance energy transfer (FRET) a popular signaling mechanism. However, nonspecific folding induced by mono- or divalent metal ions can also reduce the end-to-end distance and thus lead to false positive results. In this study we used a FRET pair labeled Hg(II) binding DNA and monitored metal-induced folding in the presence of various cations. While nonspecific electrostatically mediated folding can be very significant, at each tested salt condition, Hg(II) induced folding was still observed with a similar sensitivity. We also studied the biophysical meaning of the acceptor/donor fluorescence ratio that allowed us to explain the experimental observations. Potential solutions for this ionic strength problem have been discussed. For example, probes designed to signal the formation of double-stranded DNA showed a lower dependency on ionic strength.     

Rational Design of Ratiometric Fe3+ Fluorescent Probes Based on FRET Mechanism

As the most abundant transition metal element in mammals, iron(Fe) plays a vital role in life activities. It is of great significance to study the variation of Fe³⁺ level in living organisms. In virtue of the advantages of high sensitivity, good selectivity and low damage to living systems, the fluorescence detection of Fe³⁺ has attracted much attention. Compared with the intensity-based fluorescent probe, the ratiometric fluorescent probe has less interference of environmental and can realize quantitative detection. In this study, four ratiometric Fe³⁺ fluorescent probes, R1, R2, R3 and R4_, were designed and synthesized using fluorescence resonance energy transfer(FRET) mechanism to achieve quantitative detection of Fe³⁺. In the FRET systems, 1,8-naphthalimide fluorophore derivatives were adopted as donors while rhodamine B derivatives were selected as receptors. The connection sites of the donor and acceptor in R3 and R4 are different from those in R1 and R2. All the four probes showed good response and selectivity to Fe³⁺. The energy transfer efficiencies of R3 and R4 were obviously higher than those of R1 and R2. This work provided a promising strategy for the development of fluorescent ratiometic Fe³⁺sensors.     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

Building‐Block Approach for the Straightforward Incorporation of a New FRET (Fluorescence‐Resonance‐Energy Transfer) System into Synthetic DNA

The synthesis of the two new phosphoramidites 5 and 8 bearing a carbostyril (=quinolin‐2(1H)‐one) chromophore used as donor entity in our recently developed new FRET (fluorescence‐resonance‐energy transfer) system is described (Schemes 1 and 2) The high stability of the chromophore to basic conditions enables the incorporation of the phosphoramidites directly into DNA during solid‐phase synthesis (Schemes 3 and 4). Since this is also possible for the (bathophenanthroline)ruthenium(II) complex used as acceptor (Scheme 4, Steps d and e), the whole labelling procedure to insert the FRET system into synthetic DNA is straightforward and represents a major improvement to our previous strategy.     

A Micellar Complex of a Conjugated Polyelectrolyte for Efficient FRET to Dye‐Labeled DNA

A polymer–surfactant micellar complex has been studied as a fluorescence resonance energy transfer (FRET) donor to fluorescein‐labeled DNA (ssDNA‐Fl). In water, the molar absorptivity and fluorescence quantum efficiency of cationic poly(fluorene‐co‐phenylene) (c‐PFP) are substantially increased in the presence of non‐ionic surfactants. A TEM microscopic study shows the formation of a nanowire micellar complex of c‐PFP and the surfactants. About a 400% enhancement of the FRET signal is measured in c‐PFP/ssDNA‐Fl with Brij 30, relative to that without surfactants. The signal amplification is successfully modulated using different types of non‐ionic surfactants which perturb the complexation, fine‐structure of the complex (i.e., donor‐acceptor separation), and the resulting energy transfer process.

     

Verification and Biophysical Characterization of a New Three‐Color Förster Resonance‐Energy‐Transfer (FRET) System in DNA

We report on a new three‐color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin‐2(1H)‐one)‐derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A₁, and a Cy dye as A₂ (FRET=Förster resonance‐energy‐transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time‐resolved mode. The FRET system was established on double‐stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules.     

Dynamics of Fluorescent Dyes Attached to G‐Quadruplex DNA and their Effect on FRET Experiments

FRET spectroscopy is a promising approach for investigating the dynamics of G‐quadruplex DNA folds and improving the targeting of G‐quadruplexes by potential anticancer compounds. To better interpret such experiments, classical and replica‐exchange molecular dynamics simulations and fluorescence‐lifetime measurements are used to understand the behavior of a range of Cy3‐based dyes attached to the 3′ end of G‐quadruplex DNA. The simulations revealed that the dyes interact extensively with the G‐quadruplex. Identification of preferred dye positions relative to the G‐quadruplex in the simulations allows the impact of dye–DNA interactions on FRET results to be determined. All the dyes show significant deviations from the common approximation of being freely rotating and not interacting with the host, but one of the Cy3 dye analogues is slightly closer to this case.     

吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒的制备及其性质研究

以蛋白质或多肽修饰的吲哚类菁染料Cy3为内核, 采用实验条件简单的油包水反相微乳液方法成核, 通过正硅酸乙酯水解形成的网状二氧化硅包壳的方法制备吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒. 考察了以不同等电点的蛋白质和多肽修饰的Cy3为内核材料对吲哚类菁染料Cy3嵌入的核壳荧光纳米颗粒制备的影响. 结果表明, 分别采用人免疫球蛋白(IgG)或多聚赖氨酸修饰的Cy3为内核材料, 都能制备荧光强度高、荧光稳定性强和染料泄漏极少的Cy3嵌入的核壳荧光纳米颗粒. 进一步对Cy3嵌入的核壳荧光纳米颗粒进行了表征, 并将基于这一新型的荧光纳米颗粒建立起来的生物标记方法初步应用于流感病毒DNA的检测, 其检测线性范围为3.18×10-10～1.27×10-9 mol/L, 检测下限为3.51×10-10 mol/L, 相关系数r为0.986 5.