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1 Introduction

1.1 DNA and Ru(bpy)2dppz2+

Ru(bpy)2dppz2+ is an organometallic molecule (Figure 1.1), centered around a Ru atom bound with three ligands, whose one of them dipyridophenazine (dppz) allow for its strong interaction with DNA: it intercalates between DNA base pair. It exhibits the unique property of being luminescent when intercalation occurs. As a consequence, it plays an important role in exploring the structure and properties of DNA. It can be seen as a reporter of DNA structure, so it is very interesting to study on the interaction between the Ru(bpy)2dppz2+ and DNA.

Firstly, a general overview of DNA, structure and physical properties, is presented in the introductory part. Secondly, complexation of ruthenium and DNA is introduced in introduction.

NNNNNNNNNRuNNNbpy + phz = dppz[Ru(bpy)2dppz]2+

Figure 1.1: Chemical structure of ligand (dppz) fragments and [Ru(bpy)2dppz]2+.

1.2 Deoxyribonucleic acid (DNA)

DNA is a very long, threadlike macromolecule built form a large number of dexoyribonucleotides, each composed of a base, a sugar, and a phosphate group. The bases of DNA molecules carry genetic information, whereas their sugar and phosphate group perform a structural role. All living cells on Earth, without any known exception, store their hereditary information in the universal language of DNA sequences. These monomers string together in a long linear sequence that encodes the genetic information.

1.2.1 Chemical structure of DNA

Fi

gure 1.2: (A) Building block of DNA. (B) DNA strand. (C) Templated polymerization of new strand. (D) Double-stranded DNA. (E) DNA double helix [1].

DNA is made up of simple subunits, called nucleotides (Figure 1.2A and Figure 1.4), each consisting of a sugar-phosphate molecule with a nitrogen-containing sidegroup, called base, attached to it [2]. The bases are of four types (adenine, guanine, cytosine, and thymine), corresponding to four distinct nucleotides, labeled A, G, C, and T, the chemical structure of these bases is shown in Figure 1.3. A single strand of DNA consists of nucleotides joined together by sugar-phosphate linkages (Figure 1.2B). Note that the individual sugar-phosphate units are asymmetric, giving the backbone of the strand a definite directionality, or polarity. The backbone has two important features: it is highly flexible and is highly charged (in water, at room temperature). The negative charge of the backbone is due to the fact that the phosphate groups in water or under physiological pH are fully dissociated. Through template polymerization (Figure

1.2C), the sequence of nucleotides in an existing DNA strand controls the sequence in which nucleotides are joined together in a new DNA strand; T in one strand pairs with A in the other, and G in one strand pairs with C in the other. The new strand has a nucleotide sequence complementary to that of the old strand, and a backbone with opposite directionality, i.e. GTAA. . . of the original strand, and . . .TTAC in complementary one. Normally DNA molecule consists of two complementary strands (Figure 1.2D). The nucleotides within each strand are linked by strong (covalent) chemical bonds; the complementary nucleotides on opposite strands are held together more weakly, by hydrogen bonds. The two strands twist around each other forming a double helix (Figure 1.2E)--a strong structure that can accommodate any sequence of nucleotides without changing its basic structure. The bases can pair in this way only if the two polynucleotide chains that contain them are anti-parallel to each other.

NH2NHCCCCNCNHHH2NHNCOCCCNCNHHNNAdenine(A)Guanine(G)OHNCCCCCH3NOCNH2CCCHONHHNHHThymineCytosine(T)(C)

Figure 1.3: Chemical structure of four types of the DNA bases.

Figure 1.4:: DNA consists of nucleotides. Single nucleotide is a sugar-phosphate molecule with attached nitrogen-containing base. Here, thymine (T) is presented [1].

1.2.2 Physical properties of DNA

The physical structure of double-stranded DNA is determined by the fact that its character is amphiphilic [3]. That means that one part of DNA chain (the phosphate backbone) is hydrophilic and another one (bases) is hydrophobic. Along with the flexibility of backbone, this amphiphilic character is a cause of double-helical structure of DNA. Double-stranded DNA occurs as a ladder which is twisted around its axis right-handed. The diameter of such twisted double-helix is 2.37 nm [3]. The twisting angle between adjacent base pairs is 34.6° and the distance between two neighbor nucleotides is 0.34 nm. The number of base pairs coinciding with the full twist (360°) of DNA double-helix is around 10.4 [3]. That full twist repeats itself in every 3.4 nm (Figure 1.5). Between two molecules of deoxyribose attached to complementary base pairs, there is a space for creating grooves, which go along the whole DNA chain. Both

Figure 1.14: Steady-state emission spectra of Ru(bpy)2(dppz)2+(10 ??) in the absence and presence of B-form (top left), Z-form (top right), and A-form (bottom) double-helical DNA [36].

2). Ru(bpy)2dppz2+--the light switch effect as a function of nucleic acid sequence and conformation [37]:

The spectroscopic propeties for Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ on binding to nucleic acids of different sequences and conformations have been explored by spectroscopic measurements. Both complexes (Ru(bpy)2dppz2+ and Ru(phen)2dppz2+) serve as “molecular light switch” for DNA, luminescing intensely in the presence of DNA but no photoluminescence in aqueous solution. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; the metal complex, upon intercalation into the DNA helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap

with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, these two complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ can be seen as unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA. 3). Sensitivity of Ru(bpy)2dppz2+ luminescence to DNA defects [50]:

Ru(bpy)2dppz2+, upon binding to DNA contained a defect, exhibits significant luminescent enhancements above then well matched DNA. In the presence of a single base mismatch, large luminescent enhancements are evident when ruthenium bound to an oligonucleotide containing an abasic site (Figure 1.15). Titrations with hairpin oligonucleotides containing a variable mismatch site exists correlation between the level of luminescent enhancement and the thermodynamic destabilization associated with the mismatch. This correlation is reminiscent of that found earlier for a bulky rhodium complex that binds mismatched DNA sites through metalloinsertion, where the complex binds the DNA from the minor groove side, ejecting the mismatched bases into the major groove. This metalloinsertion mode for the dppz complex at the defect site is proved by differential quenching studies with minor and major groove quenchers and time-resolved emission studies. For sure, the utility of Ru(bpy)2dppz2+ can be seen as a sensitive reporter of DNA structure with defect site.

Figure 1.15: Titrations of Ru(bpy)2dppz2+ with DNAs containing defects. Top: DNA sequences of matched, mismatched and abasic 27-mer duplex DNA (R denotes a tetrahydrofuranyl abasic site). Bottom: plots of the integrated emission intensity (λex = 440 nm) of rac- (left), Δ- (middle), and Λ-Ru(bpy)2dppz2+ (right) (100 nM) upon increasing the concentration of DNA in 50 mMNaCl, 5 mM Tris, pH7.5 [50].

4). Crystal structure of ?-Ru(bpy)2dppz2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation [11]:

The versatile binding modes attainable for octahedral metal complexes bearing an intercalating ligand are depicted in detail. Here it is shown that two independent views of metalloinsertion in this work, two of intercalation and one of end-capping (Figure 1.16 and 1.17). The metal complex binds with DNA through metalloinsertion in the minor groove at destabilized regions of the DNA, accompanied by extrusion of the mismatched bases. This binding mode has been observed previously with a sterically expansive ligand, but this structure clearly demonstrates that a narrower ligand such as dppz is equally capable of recognizing mismatches by the means of metalloinsertion, pointing to the generality of this binding mode. The smaller size of the dppz ligand also allows the ruthenium complex to bind through classical intercalation between two consecutive well-matched base pairs. Curiously, intercalated complexes are also located in the minor groove, which they hypothesize is stabilized by extensive ancillary interactions. This discrepancy notwithstanding, the

crystal structure attests to the remarkable structural flexibility of DNA upon high-density ligand binding, illustrates the nuanced binding geometries sampled by a non-covalently bound small molecule, and highlights the dominance of metalloinsertion as the preferred binding mode to destabilized regions of DNA. These newly obtained structural understandings will help guide the development of future generations of metal complexes as chemical tools and medicinal agents.

Figure 1.16: Structure of ?-[Ru(bpy)2dppz]2+ bound to the mismatched oligonucleotide 5′-CGGAAATTACCG-3′. Front view (left) and view rotated 90 degrees (right) around the helix axis. Three DNA-binding modes are observed: (1) metalloinsertion, whereby the ruthenium complex (red) inserts the dppz ligand into the DNA duplex (grey) at the mismatched sites through the minor groove, extruding the mispaired adenosines (blue); (2) metallointercalation, whereby the complex (green) binds between two well-matched base pairs; (3) end-capping, whereby the complex (yellow) stacks with the terminal Watson–Crick pair of the duplex [11].

Figure 1.17: The end-capping complex. The duplex (dark grey) is end-capped by the ruthenium complex (red), which stacks between an extruded adenosine (blue) and the first complex (yellow) in a crystallographically related duplex (light grey). The last GC base pair (cytidine, cyan; guanosine, green) forms a frayed end [11].

1.5 Characterization of Ru(bpy)2dppz2+ and DNA interaction

Ru(bpy)2dppz

2+

exhibits intense luminescence when upon binding to DNA due to

the ligand part (dppz) intercalation into intact DNA base pair. There are two very interesting questions which deserve to be determined by their interactions as the interaction between them reaches equilibrium, these will help us to understand the interaction modes and the structural changes of DNA. 1) Determination of affinity constant (Ka)

Ru?DNAbpKa[Ru?DNAbp]

A series of titrations are performed in our study, the luminescence intensity

increases with the increase of ruthenium concentration (CRu), there exists linear

relationship between luminescence and rate of complexation at lower ruthenium concentration, which will be presented in the experimental part of chapter 2. In order to compute the affinity constant and complexation degrees without any hypothesis, in particular without assuming that the concentration at which DNA is saturated is known, a analysis method given by C. J. Halfman and T. Nishida is employed to calculate them according to the luminescence intensity change induced by Ru(bpy)2dppz2+ intercalation [52]. The values of affinity constant (be of the order of 106 M-1 at [NaCl] = 10 mM) gotten from Nishida method is similar with that value computed from the other scientists. 2) Dynamical changes of DNA helix

Although it is well known that the length of DNA increases approximately, when

one Ru(bpy)2dppz2+ molecule intercalates into DNA base pair [53]. The induced dynamical changes, in terms of DNA, fluctuations are not established. In Chapter 3 of this manuscript, we will quantify the change of flexibility induced by the intercalation of Ru(bpy)2dppz2+, of a short dsDNA (15 base paris long). What about the kink when the binding of DNA with Ru(bpy)2dppz2+ occurs?

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