File Name: a b and z form of dna .zip
Three major forms of DNA are double stranded and connected by interactions between complementary base pairs. Bases fit in the double helical model if pyrimidine on one strand is always paired with purine on the other. This pairs a keto base with an amino base, a purine with a pyrimidine.
NCBI Bookshelf. The double-helical structure of DNA deduced by Watson and Crick immediately suggested how genetic information is stored and replicated. As was discussed earlier Section 5.
Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA.
Nucleic acids were first discovered as vectors of heredity in living organism and they are now also known to be involved in a variety of cellular processes like, for example, transcription, regulation and biocatalysis. Since these structures are involved in important cellular processes, the regulation of which can be key in human diseases such as cancer 4 , it is of utmost importance to understand their structural properties, thermodynamics and mechanisms of formation.
Its crystal structure was first reported in by the team of Alexander Rich and its biological relevance has long been debated, but it is now admitted that left-handed DNA and RNA structures can be found in vivo and play a role in transcription regulation 5—7.
Compared to B-DNA, Z-DNA is thermodynamically disfavored under normal cellular conditions and it forms only under specific conditions such as high salt concentrations or negative supercoiling, although the exact mechanism of its formation remains to be determined 8— To screen for the increased electrostatic repulsion of the phosphate-containing backbones that are closer to each other in Z-DNA than in B-DNA, high salt concentration is needed for in vitro studies.
However, recent evidence indicates that anions can also influence B- to Z-DNA transition and so a pure electrostatic model fails to accurately explain the role of salts in Z-DNA formation Monitoring of the B- to Z-DNA transition has up until now relied almost exclusively on circular dichroism CD measurements, since the switch from a right- to a left-handed helix results in a very distinctive inversion of the signs of the CD peaks 10 , 19— However, NMR and CD both require large amounts of sample and X-ray diffraction is limited to crystallized, solid state structures and therefore makes it impossible to study the dynamics of the system in solution.
Fluorescence is a very sensitive and versatile technique that allows monitoring of real-time processes and is, thus, particularly suited to study nucleic acids structure and dynamics 17 , However, utilizing fluorescence spectroscopy requires adequate probes able to monitor the differences between the B- and Z-forms of DNA. The golden standard of fluorescent base analogues, 2-aminopurine 2-AP , has been used to study the base-pair extrusion at B-Z junctions, taking advantage of its environment-sensitive emission 23 , While 2-AP remains one the most widely used fluorescent base analogues, it is a fairly poor fluorophore once incorporated into DNA and its use in the field of Z-DNA seems limited to the study of B-Z junctions and cannot be extended to a more comprehensive study of the Z-DNA structure.
While such an investigation gives highly valuable insight on the system dynamics, single-molecule fluorescence is still not a routine experiment in most laboratories and is not easily implemented. Hence there is a strong need for a versatile fluorescent tool that enables more flexible labelling of any part of the DNA sequence and that reliably monitors the B- to Z-DNA transition. As a consequence, the FRET efficiency will not only depend on the distance between the acceptor and the donor but also significantly on their relative orientation.
This makes it possible to use FRET quantitatively to resolve solution structures 28 and even small modifications of the DNA conformation may result in significant changes in the FRET efficiency for these probes. Three different base pair separations 4, 6 and 8 were investigated for both structures. Moreover, we show that the fluorescence intensity variation can be monitored over time to measure rate constants of the salt-induced B- to Z-DNA transition.
Importantly, we find that despite their extended aromatic surfaces the modified tricyclic cytosines only have minor influence on the transition compared to unmodified DNA. This suggests that the tricyclic cytosines can be used efficiently and reliably in standard ensemble fluorescence experiments to monitor B- to Z-DNA transition virtually without interfering with the nucleic acid system structure and dynamics.
DNA sequences investigated. Position of donor, tC O , in red and acceptor, tC nitro , in green. Cytosines marked by a star are methylated unless they are replaced by tC nitro. In the hemimethylated sequence the donor strand containing tC O is not methylated.
The red box marks expected position of the extruded base pair according to literature 16 , The B-DNA structure is shown only for the purpose of comparison. The structures of the modified cytosines were imported into Chimera and superimposed onto the native cytosines at the desired positions.
Top and side view of the B- and Z-DNA structures showing the positions and orientations of tC O red and tC nitro green for different base-pair separations. In the top view, transparency increases with the number of base pairs separating tC O and tC nitro. Kinetics of the B- to Z-DNA transition were measured by following the evolution of the CD signal at nm or of the fluorescence at nm immediately after the addition of B-DNA to the high salt buffer over a period of to s.
To evaluate the feasibility of using the tricyclic cytosine FRET-pair, we first calculated theoretical values of FRET-efficiencies for different base-pair separations and compared them to the corresponding, previously known values in B-DNA. In this way the DNA conformations can be experimentally determined with steady-state and time-resolved fluorescence spectroscopy using the FRET pair and with CD as a reference method.
We used it to model the three different base-pair separations 4, 6 and 8 that we have in our GC 7 hairpin. As can be seen in Table 1 , the high FRET anticipated for the four base-pair separation in B-form is not expected to change significantly due to the very short distance. The hairpin sequence Figure 1 built up by GpC repeats, with the exception of the 4 thymine loop, is expected to undergo a total conversion from B- to Z-conformation at high salt concentration.
To study the effect of salt on the transition, two different high sodium chloride concentrations 3 and 4. A few minor differences in intensity were observed between the unmodified reference sequence and the corresponding modified ones, which most likely originate from the difference in absorptive properties between the native cytosines and the tricyclic cytosines that replace them.
Also at this salt condition the CD spectra are virtually identical for the native and the corresponding modified oligonucleotides. Hence the data strongly suggest that the hairpin sequence undergoes the expected B- to Z-DNA transition, whether the sequence contains our modified tricyclic cytosines or not.
A salt titration from 2 to 4. CD spectra and FRET efficiencies calculated from time-resolved emission measurements of the GC 7 hairpin sequence at different salt concentrations. To facilitate comparison, the CD spectra of the reference structures at the three salt conditions are displayed together in panel D. E FRET efficiencies calculated from time-resolved measurements of the GC 7 hairpin structures at the three salt conditions. Measurements performed at room temperature in pH 7.
The intrinsic fluorescence of tC O increases upon B- to Z-DNA transition and this is accompanied by a change of fluorescence lifetime from 3. Recently, a fluorescent guanosine analogue was reported that, in a similar fashion, was able to monitor B- to Z-DNA transitions with a fluorescence intensity increase This increase in emission, thus, appears to be an intrinsic property of Z-DNA regardless of the fluorophore. FRET experiments have significant advantages compared to intensity-based measurements since environmental solvents, cosolutes, oxygen and instrumental factors lamp fluctuations, detector sensitivity influencing fluorescence intensity, in particular when working in complex samples and media, can be handled more reliably.
This means that the additional donor intensity change upon addition of the acceptor is a change strictly associated with the FRET mechanism and not to other external factors. The advantage of FRET is particularly large when using time-resolved emission, since measurements of fluorescence lifetimes are independent of concentration and therefore also avoid such experimental errors that can accompany intensity-based studies.
The time-resolved FRET efficiencies measured here were calculated with high reproducibility in duplicate from the measured average lifetime values and are shown in Figure 3E and reported in Table 2 see Table S3 in Supplementary Material for fluorescence decay fittings and lifetime values. FRET-efficiencies calculated from steady-state emission spectra are virtually similar to those from the time-resolved measurements but experimental errors are higher Figure S4 and Table S1 in Supplementary Material.
The general trend and the values correlate well with the theoretical calculations Table 1 , except for the four base-pair separation sequence sample D3A8 , where an unexpected change in FRET efficiency is observed. The larger variation in the theoretical estimates of the FRET-change in the 6 base-pair separation case compared to experiment could be an effect of the very high responsiveness to small structural variations around such base-pair separations A minor stabilizing effect of the Z-form of DNA by the tC-bases was observed but it does not interfere with the measurements when the salt effect is strong enough to induce complete conversion to the Z-form 4.
However, at 3 M NaCl where the unmodified sequence is present in a mixture of B- and Z-form DNA, the additional stabilization of the tC-bases in the modified oligonucleotides is clearly visible Figure 3B. The sequence is comprised of a methylated or hemimethylated GC 5 repeat segment susceptible of forming Z-DNA and of an 8 bp segment expected to remain in the B-form even at high salt conditions Figure 1.
The B-DNA part of the sequence was selected randomly, except for the three AT base pairs which have been proven to be necessary for the formation of B-Z junctions as they are weaker and, thus, can induce the flexibility necessary for the DNA sequence to switch from a right- to a left-handed helix 15— The short distance sample, D5A10, was only studied in its methylated form since, for similar reason as in the hairpin structure, at this base-pair separation it is not expected to give useful data in the FRET measurements.
The reference sample in panels A to C is the sequence containing no tricyclic cytosine molecules. However, the ellipticity at nm remains negative at 4. The differences between the CD-spectra of the reference and the modified sequences at both low and high salts are larger than in the case of the hairpin sequence Figure 3A , B. Also here the variations most likely originate from differences in absorption between normal C and the tricyclic cytosines.
Additionally, unlike for the hairpin structures, excess single strand can here contribute to the CD and increase the differences. Due to the significant differences in the CD spectra, salt titrations from 2 to 4. These results confirm that despite the differences in intensity, the reference and modified sequences undergo a similar change of conformation and end up with the same BZ junction structure. Again, the FRET-efficiencies calculated from steady-state emission spectra are virtually similar to those from the time-resolved measurements but experimental errors are higher Figure S4 and Table S1 in Supplementary Material.
Moreover, similar steady-state FRET results were observed for the methylated sequence D5A12 at a sample amount times lower than for the CD experiments, without changing the measurement conditions data not shown. This highlights the advantage of fluorescence over CD: a satisfactory CD spectrum requires a significantly higher sample amount the amount used in the CD-experiments presented in this study is approaching the limit and a longer acquisition time in our case we used 12 min for CD versus 3 min for fluorescence.
It is worth noting that the intrinsic fluorescence intensity and lifetime of tC O remain much more stable during this transition than in the case of the hairpin for spectra and lifetimes values see Figure S3, Table S4 and S5 in Supplementary Material. This results in a different relative orientation of tC O and tC nitro , and thus in a different orientation factor and consequently FRET efficiency, in the B-Z DNA junction compared to in the hairpin vide supra where the structure is all Z-form.
The trend in the FRET efficiency variation is similar to that of the hairpin, with an increase for the 6 bp separation duplex and a decrease for the 8 bp separation duplex. As expected, the structures and FRET-efficiencies at mM are virtually identical for the methylated and hemimethylated sequences. However, under high salt conditions the CD-spectra of the hemimethylated sequences show that the transition occurs to a significantly smaller extent than for the fully methylated one Figure 4C and Table 3.
The negative band at nm is indeed less pronounced than for the fully methylated sequence. This emphasizes the well-known ability of 5-methylcytosines to facilitate the B-Z transition.
The effect of methylation is clearly shown by comparing the CD-spectra of the unmethylated, hemimethylated and fully methylated unmodified sequences at different salt concentration Figure 4D. The large decrease in intensity visible in the long-wavelength part of the CD spectrum of the unmethylated sequence when increasing the salt concentration BZ sequence, Figure 4D has been observed previously and seems to be a consequence of the presence of high salt B-DNA structures Importantly, the absence of significant change in CD signal in the nm band for the same sample is a clear indication that no Z-DNA is formed The need for cytosine methylation and the absence of B- to Z-transition at 3 M NaCl data not shown shows that the transition is thermodynamically more disfavored compared to the same transition in the full Z-DNA hairpin due to the necessity of forming a B-Z DNA junction.
At high salt the variation of the efficiencies of the hemimethylated samples follows the same trend as for their methylated counterpart but with a lower magnitude. Despite the slight stabilizing effect of the tC-bases observed in the hairpin structure, the hemimethylated sequences all seem to display similar amount of Z-DNA regardless of the modifications, although exact quantification is not possible due to the mixture of B- and Z-DNA.
Along with the salt titrations performed on the methylated sequences see Supplementary Material Figure S1 and S2 , this demonstrates that the stabilizing effect of the tC bases observed in the case of the hairpin is negligible compared to that of cytosine methylation. This property is necessary if the tC bases should serve as virtually non-perturbing probes for Z-DNA and was not obvious considering that the extended ring system of the tricyclic cytosines represents a much larger structural modification to the cytosine scaffold than methylation.
For instance, C8-arylguanine have been shown to drastically stabilize Z-DNA, thus enabling the transition to occur under physiological conditions and a similar effect of the tC-probes would not have been surprising 38 , This was not the case for the unmethylated GC-hairpin investigated above.
After demonstrating the ability of our FRET pair to correctly discriminate between the B- and Z-forms of DNA, we set out to study the kinetics of the B- to Z-DNA transition in the GC 7 hairpin sequence by following the evolution of the fluorescence signal at the two high salt conditions 4. Despite being thermodynamically more disfavored, the transition in the B-Z DNA junction sequence occurred much faster, possibly due to the shorter GC-repeat segment, and was not possible to measure accurately without using stopped-flow techniques data not shown.
Since following the change in the intrinsic fluorescence of tC O during the transition vide supra is enough to obtain kinetics curves, the presence of our FRET acceptor tC nitro is not mandatory here. However, studying the kinetics for the sequences containing our complete FRET pair was also interesting in order to evaluate the effect of the modified nucleobases on the B- to Z-DNA transition. Also, the signal variation during the transition is significantly higher in the case of the 8 base-pair separation FRET-sequence than the intrinsic variation of tC O fluorescence intensity, thus affording more reliable experimental data.
Measurements were performed at 4. Lines are mono-exponential fits of the experimental data. In all cases a significant variation in fluorescence was observed due to the change in FRET efficiency or as an effect of the change of the intrinsic fluorescence of tC O D3. The kinetics curves could be well fitted to a mono-exponential function Figure 5 , suggesting first-order kinetics for the B- to Z-transition of the hairpin.
Even though strict comparison is impossible since the rate of transition will depend on the length of the GC-segment and on the conditions used to induce the transition, the values found herein are reasonable when comparing to the previously obtained results. We compared our fluorescence kinetics measurements to investigations made by CD which currently is the most common method to follow the B- to Z-DNA transition kinetics
NCBI Bookshelf. Vladimir N. Potaman and Richard R. Also discussed are the requirements for the formation of alternative DNA structures, as well as their possible biological roles. The formation of non-B-DNA within certain sequence elements of DNA can be induced by changes in environmental conditions, protein binding and superhelical tension. Several lines of evidence indicate that alternative DNA structures exist in prokaryotic and eukaryotic cells.
In addition, the DNA may be able to exist in other forms of double helical structure. These are A and C forms of double helix which vary from B- form in spacing between nucleotides and number of nucleotides per turn, rotation per base pair, vertical rise per base pair and helical diameter Table 5. It has antiparallel double helix, rotating clockwise right hand and made up of sugar- phosphate back bone combined with base pairs or purine-pyrimidine. The base pairs are perpendicular to longitudinal axis of the helix. The base pairs tilt to helix by 6.
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There are three major families of DNA helices: A-DNA, B-DNA and Z-DNA. The helical structure of DNA is variable and depends on the sequence as well as the.
Whether this bizarre form of DNA existed in cells and had any function, and what that might be, was hotly debated for nearly half a century. But research has recently confirmed its biological relevance. So-called Z-DNA is now thought to play roles in cancer and autoimmune diseases, and last year scientists confirmed its link to three inherited neurological disorders. Today, molecular biologists are beginning to understand that certain stretches of DNA can flip from the right- to the left-handed conformation as part of a dynamic code that controls how some RNA transcripts are edited. The hunt is now on to discover drugs that could target Z-DNA and the proteins that bind to it, in order to manipulate the expression of local genes.
Methylation of cytosine at the 5-carbon position 5mC is observed in both prokaryotes and eukaryotes. In humans, DNA methylation at CpG sites plays an important role in gene regulation and has been implicated in development, gene silencing, and cancer. In addition, the CpG dinucleotide is a known hot spot for pathologic mutations genome-wide. CpG tracts may adopt left-handed Z-DNA conformations, which have also been implicated in gene regulation and genomic instability. Methylation facilitates this B-Z transition but the underlying mechanism remains unclear.
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Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism.Reply
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It is a left-handed double helical structure in which the helix winds to the left in a zigzag pattern, instead of to the right, like the more common B-DNA form.Reply
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