Study of the influence of metal ions on tRNA(Phe) thermal unfolding equilibria by UV spectroscopy and multivariate curve resolution

The influence of metal ions (Na(+), Mg(2+) and Cd(2+)) on the thermal unfolding of phenylalanine transfer ribonucleic acid (tRNA(Phe)) was studied by UV spectroscopy-monitored melting experiments. Absorbance data were obtained during the unfolding process in the range 220-340 nm and later analyzed by a multivariate curve resolution approach (MCR-ALS) based on factor analysis. This procedure determines the number of spectroscopically distinct conformations present during the unfolding process and reveals their concentration profiles and pure spectra, without any initial assumption having to be made about the number of steps in the unfolding pathway. From the concentration profiles and pure spectra, information such as T(m) values can be recovered. The results were compared with those obtained previously in spectroscopic and calorimetric unfolding experiments, showing that the multivariate approach recovers information that complements that obtained in traditional spectroscopic melting experiments.     

溶液中d(AT)_6的构象转化

 本文报道了溶液中d(AT)_6构象研究的结果。探讨了d(AT)_6的物化特征。d(AT)_6在0.05mol/L NaCl和4.5 mol/L NaCl中的UV和CD谱是典型的Z-和B-型DNA谱,UV熔化曲线形状依赖于盐的浓度。当盐浓度低于0.05 mol/L NaCl时,曲线有负斜率;而盐浓度大于0.05mol/L NaCl时,曲线的斜率变为正,即随盐浓度增加,T_m值增加。在链浓度的依赖性基础上,计算了两种构象的△H和△S。     

Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine

2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems. To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments. The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T. Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS). This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them. Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated. At pH near 4, the majority of species shows C protonated and P deprotonated. Finally, at pH values near 3, the majority of species shows both protonated C and P. These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence including the P x T mismatch, only one transition was observed at neutral pH. However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.     

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.     

Conformational study on ketamine by circular dichroism and ultraviolet spectroscopy

Since the enantiomers of the N‐methyl‐D ‐aspartate (NMDA) receptors antagonist ketamine have different pharmacological profiles, CD and UV spectroscopy were applied for the study of conformer equilibrium and pH dependence in ketamine solutions. The assignment of the configurations and conformations was performed on the basis of the “octant rule” and UV spectra. In accord with published data, it was established that, on protonation, the phenyl group of the ketamine molecule occupies an axial position, while for the base form, the ratio of conformers containing axial/equatorial aryl moieties is strongly solvent‐dependent. The CD and UV spectra indicate the presence of an intramolecular H‐bond C=O····H—N in the conformer with axial aryl moiety. Chirality 11:280–285, 1999. © 1999 Wiley‐Liss, Inc.     

Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor

Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.     

Acid-base and copper (II) complexation equilibria of poly(inosinic)-poly(cytidylic)

The acid-base and copper (II) complexing properties of the heteropolynucleotide poly(inosinic)-poly(cytidylic) acid were studied by means of potentiometric, spectrophotometric, CD, and molecular fluorescence titrations in a working aqueous medium of 0.15 M ionic strength at 37°C. The study of the acid-base equilibria has proved the existence of four different species in the pH interval 2–11. Two new species are detected in the presence of copper(II) ion, at acidic and neutral pH values. Spectrophotometric, CD, melting, and fluorescence data were treated with a multivariate curve resolution procedure that allowed the determination of the number of species simultaneously present in each system [acid-base equilibria, copper(II) complexation equilibria], and the estimation of their concentration profiles and of the pure spectra for each species. From the concentration profiles an unambiguous assignment of which species are present at any pH conditions can be made. Furthermore, the knowledge of the pure uv-visible, fluorescence, and CD spectra recovered by the proposed resolution method for each species brings important structural information. When this mathematical approach was applied to the thermal denaturation data, the melting profile and the uv pure spectra for all the different species or conformations formed in the melting process, were calculated. © 1997 John Wiley & Sons, Inc. Biopoly 42: 271–283, 1997     

Analytical characterization of the conformational transitions of polynucleotides by means of different molecular spectroscopies and multivariate curve resolution

A general procedure for the study of conformational transitions of polynucleotides is described. The equilibria between different conformations induced by salt, ethidium bromide, and temperature of poly(dG-dC). poly(dG-dC) and induced by salt and temperature of poly(A). poly(U) are investigated using molecular absorption, circular dichroism, and fluorescence spectroscopies. Spectral data obtained from experiments are analyzed by means of a factor analysis method, namely, multivariate curve resolution, which allows possible intermediate states to be detected and the pure spectra and the concentration profiles of all species present in the system to be estimated. This work shows the application of this procedure for the analysis of data matrices obtained in individual experiments but also for the analysis of several data matrices simultaneously.     

Conformational properties of DNA strands containing guanine-adenine and thymine-adenine repeats.

CD spectroscopy and PAGE were used to cooperatively analyze melting conformers of DNA strands containing GA and TA dinucleotide repeats. The 20mer (GA)10 formed a homoduplex in neutral solutions containing physiological concentrations of salts and this homoduplex was not destabilized even in the terminal (GA)3 hexamers of (GA)3(TA)4(GA)3, although the central (TA)4 portion of this oligonucleotide preserved the conformation adopted by (TA)10. This observation demonstrates that homoduplexes of alternating GA and TA sequences can co-exist in a single DNA molecule. Another 20mer, (GATA)5, adopted as a whole either the AT duplex, like (TA)10, or the GA duplex, like (GA)10, and switched between them reversibly. The concentration of salt controlled the conformational switching. Hence, guanine and thymine share significant properties regarding complementarity to adenine, while the TA and GA sequences can stack in at least two mutually compatible ways within the DNA duplexes analyzed here. These properties extend our knowledge of non-canonical structures of DNA.     

Resolution of a structural competition involving dimeric G-quadruplex and its C-rich complementary strand

The resolution of the dimeric intermolecular G-quadruplex/duplex competition of the telomeric DNA sequence 5′-TAG GGT TAG GGT-3′ and of its complementary 5′ ACC CTA ACC CTA-3′ is reported. To achieve this goal, melting experiments of both sequences and of the mixtures of these sequences were monitored by molecular absorption, molecular fluorescence and circular dichroism spectroscopies. Molecular fluorescence measurements were carried out using molecular beacons technology, in which the 5′-TAG GGT TAG GGT-3′ sequence was labelled with a fluorophore and a quencher at the ends of the strand. Mathematical analysis of experimental spectroscopic data was performed by means of multivariate curve resolution, allowing the calculation of concentration profiles and pure spectra of all resolved structures (dimeric antiparallel and parallel G-quadruplexes, Watson–Crick duplex and single strands) present in solution. Our results show that parallel G-quadruplex is more stable than antiparallel G-quadruplex. When the complementary C-rich strand is present, a mixture of both G-quadruplex structures and Watson–Crick duplex is observed, the duplex being the major species. In addition to melting temperatures, equilibrium constants for the parallel/antiparallel G-quadruplex equilibrium and for the G-quadruplex/duplex equilibrium were determined from the concentration profiles.     

Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt.

It is well known that the local conformation of a segment of DNA is dependent upon both the sequence of the segment and the conditions under which the DNA is prepared. In extreme cases, the DNA may contain regions of both right and left-handed conformations, mandating the existence of a conformational junction between the two. These B-Z junctions have been observed in plasmids but, to date, no model systems have been characterized to determine the molecular nature of these junctions. Preliminary CD, UV, and NMR studies on such a model are presented here. A 16 base pair oligonucleotide, containing a potential B-Z junction, has been synthesized and characterized by the above techniques. The results suggest that this molecule contains both right and left-handed conformations under condition of high salt, and thus a B-Z junction.     

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.     

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by ~20°C) than that of the hairpin (by ~10°C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Tetraplex DNA transitions within the human c-myc promoter detected by multivariate curve resolution of fluorescence resonance energy transfer

The nuclease hypersensitive element (NHE) III(I) of the c-myc promoter regulates the expression of oncogene c-myc and hence is an important anti-cancer target. Paranemic secondary structure formation within the promoter has been implicated in mechanistic regulation models. Here, it is shown that two monomeric tetraplexes form within the c-myc promoter, which coexist in solution. The development and application of a new experimental approach for detection of conformation transitions in nucleic acids [which exploits the sensitivity of fluorescence resonance energy transfer (FRET) for theoretical spectral resolution by multivariate curve resolution-alternating least-squares (MCR-ALS) method] has been used for this study. The pK(a) for tetraplex transitions are centered around 5.9 +/- 0.2 (between two intercalation topologies) and 6.8 +/- 0.1 (tetraplex to random coil). The presence of two tetraplexes has been further confirmed by S1 nuclease digestion. Finally, it is established that MCR-ALS analysis of FRET at different temperatures, pH, and salt concentrations allows resolution of pure species. Results are discussed in the light of recent observations implicating paranemic DNA motifs within the c-myc NHE in regulation of the oncogene. This method has several advantages over other methods vis-à-vis, high sensitivity and linear detection over a wide concentration range and, particularly, potential applications in intracellular probing.     

Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: implication for SNP genotyping

The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. − dF/dT (or dA/dT) vs T, lnK (or RlnK) vs T_M, ΔG vs T_M, ΔS vs T_M and ΔH vs T_M are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. AY151039) of chromosome 13 having either A or G mutation at position − 26.     

Synthesis and properties of DNA-duplexes, containing 9-(1'-hydroxy-2'-(hydroxymethyl)ethoxy)methylguanine]

Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.     

Intramolecular G-quadruplexes from microsatellite d(GT)12 sequence in the presence of K+

Polymorphic d(GT)n microsatellite sequences are known to drastically affect genes expression. By use of CD spectroscopy, UV melting, fluorescence polarization of EtBr probe and FRET, we detected formation of a new fold with three G-quartets by d(GT)12 oligonucleotide in 0.01 M Na phosphate buffer, pH 8.0, in the presence 0.1 M KCl. Monomolecular type of the structure was verified with measurements of rotational relaxation time (p = 28 +/- 0.5 ns) of EtBr:d(GT)12 complex. CD spectra supported G-quartets formation. A distance between FITC, covalently attached to 5'-end of d(GT)12, and intercalated EtBr molecule was estimated using FRET (R < or =17 A). These data are in agreement with the proposed self folding of d(GT)12.