Sample fluorescence is detrimental to Raman spectroscopic analysis. Several algorithms are proposed to achieve automatic fluorescence rejection (AFR) based on shifted excitation Raman difference spectroscopy. The algorithms are mathematically linear and can be automated. The methods are based on a wavelength-tunable laser and the measurement and calibration of both the Raman and the excitation spectra. Applying the AFR methods to highly fluorescent samples significantly reduces the fluorescence background and reveals weak Raman features unidentifiable using traditional methods. Fixed pattern “noise” associated with the background can be completely removed. The merits of each algorithm are discussed and the best excitation frequency shift to perform the analysis is found to be comparable to the widths of major Raman peaks.
SynopsisWe have synthesized and studied the CD spectra of five new double-stranded RNA polymers: poly [r(A-G). r(C-U)], poly[r(A-U-C).r(G-A-U)], poly[r(A-C-U)-r(A-G-U)], poly[r(A-A-C).r(G-U-U)], and poly[r(A-C-C).r(G-G-U)]. Together with previously published spectra of seven other RNA sequences, the spectra of these new sequences provide a library sufficient to approximate the spectra of all other RNA sequences by first-neighbor formulas and, in addition, give four spectra with which we may test the validity of first-neighbor approximations. (1) We find that the spectra of RNA sequence isomers are very different, but that the spectra essentially do obey first-neighbor relationships. (2) We have derived tentative first-neighbor assignments of negative bands at about 295 and 210 nm in the CD spectra. (3) A test of spectral independence shows that among the 12 polymer spectra there are a t least seven significant independent spectral shapes, one less than the eight needed to give the most accurate spectral analysis of an unknown RNA sequence for its first-neighbor frequencies. (4) Spectra are calculated for RNAs of random base composition, approximating natural RNAs having complex sequences. (5) A T-matrix of spectral components assigned to the firstneighbor base pairs is derived from 10 of the spectra. This matrix allows an estimation of the CD spectrum of any other known RNA sequence or an analysis of the spectrum of an unknown sequence for its distribution of first-neighbor base-pair frequencies. (6) Test analyses of two of the synthetic polymers and of two natural RNAs set a probable limit on the accuracy of first-neighbor frequency determinations using this T-matrix. (7) Finally, we summarize in an appendix the melting temperatures for all the RNA and corresponding DNA sequences; it appears that the T , values of both DNAs and RNAs approximately obey first-neighbor relationships.
SynopsisThe circular dichroisin spectra of many natural DNAs and double-stranded synthetic polynucleotides were obtained. The eight firstneighbor 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 nat,ural 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 f0.02 mole percent. The TG, CA, and TA first-neighbors 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 a t 260 nm and the base composition can also be calculated from the CD spectrum. INTRODUCTIONGray and Tinoco' developed the theory relating the circular dichroism (CD) spectrum for a double-stranded polydeoxyribonucleic acid (DNA) to linear combinations of contributions from the neighboring groups of units which make up the two complementary strands of the DNA. The present work was begun to test the validity of their equations in the first-neighbor approximation. A further purpose was to try to obtain as much sequence information as possible from a CD spectrum and conversely, to compute a spectrum given the appropriate sequence information.We start with a number of DNAs with known first-neighbor frequencies, extinction coefficients, and CD spectra. The contribution to the CD spectrum and extinction coefficient from each The approach used is empirical. of the independent first,-neighbors is resolved. These contributions can then be used to determine the first-neighbor frequencies and extinction coefficient for a DNA for which these parameters are unknown. EXPERIMENTAL PROCEDURES DNA PreparationsSources of DNA. It was necessary to obtain DNAs that had been analyzed for first-neighbor (or nearest-neighbor) frequencies. The bacterial strains used for the original work by Josse et a1.2 were not maint a i~i e d .~ Therefore, the sources of DNA used in this work were determined solely by convenience, the supposition being that the first-neighbor frequency distribution does not vary significantly among strains of the same species. (The crab dAT, being from a different genus, was not used in the general analysis.) A sample of Cancer antennarius light component DNA (crab dAT) was the gift of Dr. James C. Wang (Department of Chemistry, University of California, Berkeley). DNA was isolated from the five bacterial strains by a modification of 1Llarmur's procedure.5 The M . phlei cells were lysed by sonication with a model BP-1 Biosonik Oscillator and a probe attachment a t maximum power. The other bacterial cells were lysed with a combination of lysozyme (Worthington) and dodecyl sodium sulf...
SynopsisThe linear dichroism induced in a solution of electrically anisotropic molecules by a pulsed electric field has been studied. Equations have been obtained which express the dichroism as a function of dipole moment, excess polarizability, field strength, and the angle 01 between the dipole moment and the transition moment for the absorption band. These expressions have been related to the experimentally observed difference signal in such a way that when the dichroism is measured as a function of field strength the permanent moment, excess polarizability and angle a can be determined.Experiments have been carried out on tobacco mosaic virus (TMV), which is similar in its properties to the theoretical model. The polarizability anisotropy and rotary diffusion constant for the monomer and dimer of TMV have been obtained from these experiments. In addition to the molecular parameters mentioned above, the saturated electric dichroism of the virus was measured as a function of wave length and the presence of an n--a* transition in the tryptophan spectrum was indicated.Further experiments measuring dichroism as a function of pH demonstrated the general denaturation of the virits at high pH (10-11) bltt also the existence of a stable fraction which is not fragmented even at the high pH involved.
The ideal quality control method for pharmaceutical products should be capable of rapid nondestructive testing of intact tablets or capsules. Raman spectroscopy using near-infrared excitation is shown to be capable of obtaining useful spectral data directly from drug formulations in gel capsules and from the gel capsules inside blister packs. The Raman data collected from the capsules inside blister packs containing 0-100 mg of the active ingredient (bucindolol), when coupled with multivariate calibration, resulted in a calibration SEP of 3.36 mg. The largest source of error was found to be due to sample inhomogeneity. Even so, the method is shown to have significant potential as a rapid nondestructive quality control method for pharmaceutical samples.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.