A method for estimating the nearest neighbor base-pair content of RNAs using CD and absorption spectroscopy.

CD and absorption spectra are sensitive to the secondary structure of RNAs. By fitting the spectra contained in our basis set to the CD and absorption spectra of an RNA of known sequence, we could determine the fractions of base pairs, the fractions of each of the nearest neighbor base pairs, and the fractions of the single-stranded nucleotides in that RNA. The basis set included 58 CD and 58 absorption spectra. The fitting was done with a guided selection routine. The estimated error was about 0.05 for predicting the fractions of the nearest neighbor base pairs, 0.06 for predicting the fractions of A.U, G.C, and G.U base pairs, and 0.04 for predicting the fractions of the single-stranded nucleotides.     

Circular dichroism spectra of DNA oligomers show that short interior stretches of C.C+ base pairs do not form in duplexes with A.T base pairs

Circular dichroism (CD) experiments were carried out on a series of DNA oligomers to determine if short internal stretches of protonated cytosine-cytosine (C.C+) base pairs could coexist with adenine-thymine (A.T) base pairs. (1) C.C+ base pairs did form in the absence of A.T base pairs in the individual oligomers d(AACC)5 and d(CCTT)5, as indicated by the appearance of a long-wavelength CD band centered at 282-284 nm, when the pH was lowered to 6 or 5 at 0.5 M Na+. A comparison of measured with calculated spectra showed that d(CCTT)5 at pH 5, 0.5 M Na+, 20 degrees C, likely adopted a structure with a central core of stacked C.C+ base pairs and looped-out thymines. Under the same conditions, it appeared that C.C+ base pairs also formed in d(AACC)5, but with the adenines remaining intrahelical. Each of these oligomers showed a cooperative transition for formation of C.C+ base pairs as the temperature was lowered, with C.C+ base pairs forming at a higher temperature in d(CCTT)5 than in d(AACC)5. A.T base formed in equimolar mixtures of d(AACC)5 plus d(CCTT)5 as monitored by an increase in the negative magnitude of the 250-nm CD band. However, a large increase did not appear at about 285 nm in CD spectra of the mixtures, showing that there were no stacked C.C+ base pairs in the d(AACC)5.d(CCTT)5 duplex even though they formed under the same conditions in the individual strands. Thus, in this duplex, A.T base pairs prevented the formation of neighboring internal C.C+ base pairs. (2) CD measurements were also made of d(A10C4T10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

The influence of salt concentration on CD spectra of tRNA and aminoacyl-tRNA synthetase

The CD spectra of serine tRNA or seryl-tRNA synthetase were measured. The [theta] values at 210 nm were minimum at 50mM - 0.2M NaCl, at that concentration the velocity of aminoacylation was maximum. This results suggest that A . U and G . C base pairs loosened. The [theta] values at 200 nm decreased according to the decreasing of salt concentration, suggesting the decomposition of A . U base pairs. The CD spectra of seryl-tRNA synthetase at 210-240 nm were not changed in the range of 10mM-0.3M NaCl but the spectra at 260-290 nm showed minimum in the range between 50mM-0.2M NaCl. These results suggest that the influence of salt concentration on the velocity of aminoacylation depends on both the conformational changes of tRNA and seryl-tRNA synthetase.     

The ultraviolet circular dichroism of some natural DNAs and an analysis of the spectra for sequence information

The circular dichroism spectra of many natural DNAs and double-stranded synthetic polynucleotides were obtained. The eight first-neighbor contributions to the CD spectra of a DNA have been extracted from these data. Therefore, the CD spectrum for any DNA with known first-neighbor frequencies may be easily calculated. For a natural DNA the CD spectrum may be approximated by assuming the first-neighbor frequencies have the most probable values consistent with the base composition. Under favorable conditions, the measured CD spectrum can be used to determine thirteen of the sixteen first-neighbor frequencies of a DNA to ± 0.02 mole percent. The TG, CA, and TA first-neighbor cannot be unambiguously resolved by our method. The accuracy of the first-neighbor frequency analysis depends on the number of different first-neighbors present in the DNA and the extent to which they differ from the most probable value. The extinction coefficient at 260 nm and the base composition can also be calculated from the CD spectrum.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

Synchrotron radiation circular dichroism of various G‐quadruplex structures

Here we report synchrotron radiation circular dichroism spectra of various G‐quadruplexes from 179 to 350 nm, and a number of bands in the vacuum ultraviolet (VUV) are reported for the first time. For a tetramolecular parallel structure, the strongest band in the spectrum is a negative band in the VUV at 182 nm; for a bimolecular antiparallel structure with diagonal loops, a new strong positive band is found at 190 nm; for a bimolecular parallel structure with edgewise loops, a strong positive band at 189 nm is observed; and for a self‐folded chair‐type structure, the strongest band in the spectrum is a positive band at 187 nm. For the tetramolecular parallel structure, the CD signals at all wavelengths are dominated by contributions from quartets of G bases, and the signal strength is approximately proportional to the number of quartets. Our experiments on well‐characterized G‐quadruplex structures lead us to question past attributions of CD signals to helix handedness and G quartet polarity. Although differences can be observed in the VUV region for the various quadruplex types, there do not appear to be clear‐cut spectral features that can be used to identify specific topological features. It is suggested that this is because a dominant positive band in the VUV seen near 190 nm in all quadruplex structures is due to intrastrand guanine–guanine base stacking. However, our spectra can serve as reference spectra for the G‐quadruplex structures investigated and, not least, to benchmark theoretical calculations and empirical models. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 429–433, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Calculations of the CD spectrum of bovine pancreatic ribonuclease

CD spectra of bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modified form (RNase S) have been calculated, based upon high-resolution structures from x-ray diffraction. All known transitions in the peptide and side-chain groups, especially the aromatic and disulfide groups, have been included. Calculations have been performed with both the matrix method and with first-order perturbation theory. A newly developed method for treating the electrostatic interactions among transition charge densities and between static charge distributions and transition charge densities is used. The effects of local electrostatic fields upon the group transition energies are included for all transitions. Rotational strengths generated by the matrix method were combined with Gaussian band shapes to generate theoretical CD spectra. The calculated spectra reproduce the signs and approximate magnitudes of the near-uv CD bands of both RNase A and S. Agreement is most satisfactory for the negative 275 nm band, dominated by tyrosine contributions. In agreement with two previous studies by other workers, coupling between Tyr 73 and Tyr 115 is the single most important factor in this band. The positive band observed near 240 nm is dominated by disulfide contributions, according to our results. The far-uv CD spectrum is poorly reproduced by the calculations. The observed 208 nm band, characteristic of α-helices, is absent from the calculated spectrum, probably because the helices in RNase are short. A strong positive couplet centered near 190 nm is predicted but not observed. Possible reasons for these incorrect predictions of the current theoretical model in the far-uv are discussed. © 1997 John Wiley & Sons, Inc.     

The absence of non-local conformational changes in OR3 operator DNA on complexing with the cro repressor

The Interaction of the cro protein of lambda phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro. NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl] greater than 0.2 M while the whole OR3 becomes free at [NaCl] greater than 0.5 M. The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms. The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all. An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry.

Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.     

The CD of two-chain coiled coils: experiments on tropomyosin and tropomyosin segments in the tyrosine/disulfide spectral region

CD experiments are reported for several coiled-coil species in the tyrosine/disulfide (approximately 250-350-nm) region. Intact noncross-linked tropomyosin (approximately 3 degrees C) shows a negative nonsymmetric band maximal at 280 nm. This spectrum is the sum over six tyrosines/chain, and has conformational significance, since it disappears on denaturation. Experiments on an excised coiled-coil segment, each of whose chains comprise residues 11-127 of the tropomyosin sequence and only one tyrosine (Y60), reveal that not all tyrosines are alike. The spectrum at 3 degrees C shows a small negative maximum at approximately 285 nm and a substantial, hitherto unknown, positive band at approximately 270 nm, the latter masked in the parent protein by the negative contribution from the other tyrosines. A noncross-linked coiled-coil segment comprising residues 142-281, in which Y60 is absent, shows no such positive band. This peculiarity of Y60 is confirmed by absorbance spectra, with the extinction coefficient of Y60 larger in benign media than the average of the other tyrosines. Intact (3 degrees C) C190 cross-linked tropomyosin is known to yield, besides tyrosine contributions, a positive maximum at approximately 300 nm. Subtracting the corresponding data for noncross-linked tropomyosin shows that the disulfide spectrum itself actually has two equal, partly resolved bands at, respectively, 250 and 280 nm. The existence of a chiral disulfide argues for a relatively rigid, perhaps strained, local coiled coil. A C190 cross-linked segment comprising residues 142-281 shows a chiral disulfide spectrum like tropomyosin's, but another segment, comprising residues 168-284, shows none; thus removal of residues 142-167 causes loss of chirality at C190, over 20 residues away. These spectra thus contain important information on the subtle local differences in coiled-coil structures.     

CD spectra and some properties of deoxyoligonucleotide duplexes having a C:G terminus (nucleosides and nucleotides. Part 69).

In the course of an investigation of the mode of recognition of nucleotide sequences with restriction endonucleases, several deoxyoligonucleotide duplexes having G:C terminal base pairs were synthesized. The oligonucleotides having a 5'-C (3'-G) terminus showed unusual CD spectra with a negative band at the longer wavelength region, when compared to those of the same internal sequences but a 5'-G (3'-C) terminus, which showed a positive band like the B- or A-DNA type. The nature of these CD spectra was compared with those of the Z-DNAs on the effect of salt concentrations, intercalation with ethidium bromide, or 31P-NMR spectra. These unusual spectra may be attributed to the terminal effect of the 5'-C:3'-G pairs.     

Circular dichroism of double-helical oligoribonucleotides

The ultraviolet circular dichroism and absorption of 15 double-stranded helical oligoribonucleotides have been measured. These molecules of chain-length 6 to 12 contain all 10 possible nearest neighbors of Watson-Crick base pairs. They are thus good models for short double-stranded regions in RNA molecules. The contribution to the circular dichroism of each of the nearest neighbor base pairs has been obtained. The circular dichroism is found to be very sequence-dependent and may be useful in distinguishing possible secondary structures. However, the nearest neighbor approximation for circular dichroism fails to give a quantitative measure of the circular dichroism of double-strand regions.     

Circular dichroic spectra of elapid cardiotoxins

Cardiotoxins isolated from elapid snake venoms constitute a chemically homogeneous family of molecules. Within this group several biologically different subclasses exist. We report a comparative analysis of the structure of 20 cardiotoxins using circular dichroism, immunological methods and secondary-structure prediction. It is shown that cardiotoxins fall within two structural subclasses. Toxins of group I are characterized by (a) CD spectra having an intense positive band close to 192.5 nm and a negative trough at 225 nm with no positive band around 230 nm, (b) strong cross-reactivity with a polyclonal antiserum specific for Naja nigricollis toxin gamma and (c) a high tendency to form a reverse turn in the region of position 11. Toxins of group II are characterized by (a) CD spectra displaying a much weaker positive band at 192.5 nm, a negative band around 210 nm and a positive band at 230 nm, (b) little cross-reactivity with the aforementioned antiserum and (c) a high reverse-turn potential at position 31. It is suggested that the observed differences result from differing curvatures in the antiparallel beta sheet which constitutes the main secondary structure of cardiotoxins.     

The vacuum UV CD spectra of G.G.C triplexes.

Vacuum UV circular dichroism (CD) spectra were measured down to 175 nm for d(C)10, d(G)10, the d(G)10.d(C)10 duplex, and the d(G)10.d(G)10.d(C)10 triplex. A CD difference spectrum was calculated for d(G)10.d(C)10 giving the change in CD induced by forming the duplex from d(G)10 and d(C)10. The d(G)10.d(G)10.d(C)10 CD difference spectrum gave the CD induced by triplex formation from binding of d(G)10 to the d(G)10.d(C)10 duplex. In the near-UV, the d(G)10.d(C)10 and d(G)10.d(G)10.d(C)10 difference spectra resembled the difference spectrum for poly[r(G).r(C)] (Biopolymers 29, 325-333). This similarity may be an indication of similar purine base stacking. The d(G)10.d(G)10.d(C)10 vacuum UV difference spectrum had a negative band at 195 nm and a positive band at 180 nm, making it similar to difference spectra for homopolymer triplexes containing T.A.T and U.A.U triplets (Nucl. Acids Res. 19, 2275-2280). The appearance of these bands in difference spectra should be good indicators of triplex formation. The complementary oligonucleotides c-mycI d(CCCCACCCTCCC) and c-mycII d(GGGAGGGTGGGG) are part of the regulatory sequences of the human c-myc gene. G.G.C rich triplexes formed by binding c-mycII or c-mycIII d(GGGGTGGGTGGG) to the c-mycI.c-mycII duplex had CD difference spectra similar to that of d(G)10.d(G)10.d(C)10 in both the vacuum UV and near UV regions, indicating similar triplet structures.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.     

A set of nearest neighbor parameters for predicting the enthalpy change of RNA secondary structure formation

A complete set of nearest neighbor parameters to predict the enthalpy change of RNA secondary structure formation was derived. These parameters can be used with available free energy nearest neighbor parameters to extend the secondary structure prediction of RNA sequences to temperatures other than 37°C. The parameters were tested by predicting the secondary structures of sequences with known secondary structure that are from organisms with known optimal growth temperatures. Compared with the previous set of enthalpy nearest neighbor parameters, the sensitivity of base pair prediction improved from 65.2 to 68.9% at optimal growth temperatures ranging from 10 to 60°C. Base pair probabilities were predicted with a partition function and the positive predictive value of structure prediction is 90.4% when considering the base pairs in the lowest free energy structure with pairing probability of 0.99 or above. Moreover, a strong correlation is found between the predicted melting temperatures of RNA sequences and the optimal growth temperatures of the host organism. This indicates that organisms that live at higher temperatures have evolved RNA sequences with higher melting temperatures.