Oligonucleotide spectra

The progress in this area has been limited by two factors the mass range actually attained in FAB and the quantity (and purity) of compounds available. The mass range has been expanded, using, for example, postacceleration to 6 -10 kilodaltons (at 8 kV) however, very few new and larger oligonucleotide spectra have been reported. 

The conversion of the monofunctional adducts into bifunctional lesions depends drastically on the structure of the Pt drug. Obviously, Pt compounds exhibiting trans geometry form different bisadducts than cisplatin and hence, a different spectrum of antitumor activity is expected. Mechanistically, the formation and possible isomerization of bisadducts are not well understood. The assumption that hydrolysis of the second leaving group controls the formation of bisadduct may be an oversimplification. Studies with model compounds as well as with oligonucleotides have indicated that a certain nucleobase may be a powerful nucleophile toward Pt(II) if spatially in a correct position. Unfortunately, our knowledge on these interactions is at present very limited. 

Fig. 2 (a) Schematic of the formation of silver clusters using DNA oligonucleotide as scaffold. After complexation of DNA with silver cations, the mixture is reduced with NaBtL, and the fluorescent cluster is formed, (b) Absorption spectra of silver clusters acquired every 30 min using [5 -AGGTCGCCGCCC-3 ] = 10 uM. [Ag+] = 60 uM. and [BfLj-] = 60 pM. The foremost spectrum was acquired 9 min after adding the BH4, and it has at 426 nm. The inset spectrum shows the last spectmm in the series (692 min), with peaks at 424 and 520 nm. (c) Induced circular dichroism spectra. The cell path length was 5 cm. The spectra were collected 2 min (A, dashed-dotted line), 20 min (B, dotted line), 40 min (C,fine dashed line), 60 min (D, coarse dotted line), and 150 min (E, solid line) after adding the BH [32]... 

The fluorescence spectrum of the tris-acridine cryptand A-13 shows the characteristic monomer and excimer bands. Upon complexation with various organic anions (carboxylates, sulfonates, phosphates), the monomer band increases at the expense of the excimer band. The stability of the complexes depends on the contribution of the electrostatic and hydrophobic forces and on the structural complementarity. Stability constants of the complexes ranging from 103 to 107 have been measured. In particular, A-13 binds tightly to mono- and oligonucleotides, and it can discriminate by its optical response between a pyridimic and a purinic sequence. 

IR spectroscopy is extremely sensitive to RNA conformational changes and base sequence. This is illustrated in Fig. 17.1 using the IR spectra of two different RNA tetraloops. The lower panel shows IR spectra of S gcUGCGgc-d7 ( HPg ) and the upper panel corresponds to 5 -gcUCCGgc-37 ( HPC ). (The bases written in uppercase indicate the unpaired bases of the loop while those in lowercase are the bases that comprise the stem.) In each, the dashed line is the IR spectrum for the unfolded oligonucleotide (acquired at T Tm) while the solid line is for... 

Figure 2 Oligonucleotide artifacts detectable on MALDI-TOF MS. The spectrum shows a typical example of an oligonucleotide of poor quality. The presence of a sodium adduct peak is depicted (Na adduct) and evidence of depurination potentially at G nucleobases (Depurinated). The oligonucleotide sequence used was 5 OH-TAGGCCTGGGAAGCAGCA-OH 3. ...

In multiplexing, several oligonucleotides are involved. The analyte peaks in the mass spectrum for one assay must be sufficiently resolved from products of any other assay it is combined with. This includes pausing peaks. In addition, analyte peaks must fall within a specified mass window (preferably 5000-8500 Da). Using these considerations, 15 assays can be combined (Fig. 4). 

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

An ESI mass spectrum of a mixture of 1 1 d(pT)13 d(pA)14 with the gene V protein (Fig. 13a) shows the existence of a double-stranded dimer and of a complex between the protein and a single d(pT)13 oligonucleotide. Complexes of the protein with the dsDNA, or with d(pA)14 do not appear in the spectrum. This clearly points to the preference for binding ssDNA over dsDNA and poly(dT) over polyfdA). When the mixture of d(pT)13 d(pT)15 ... 

The MALDI/TOF mass spectrum of an oligonucleotide that has undergone a partial depurination reaction. Reproduced (modified) from Van Ausdall D.V. and Marshall W.S., Anal. Biochem., 256, 220-228, 1998, with permission. 

Another important advantage of mass spectrometry is its capacity to verify the incorporation of modified nucleotides. Figure 8.31 displays the mass spectrum of a synthetic oligonucleotide whose theoretical mass is 5838 Da. In addition to the desired nucleotide, other compounds are present. The mass difference between the peaks (111-151 Da) corresponds to the masses of the nucleic bases. This suggests that depurination reactions have occurred during the synthesis. 

The MALDI/TOF mass spectrum of an oligonucleotide produced by PCR amplification of a part of the gene responsible for cystic fibrosis. The top spectrum is obtained from a normal individual, whereas the central and bottom spectra are obtained, respectively, from a healthy heterozygote carrier and an ill homozygote. Reproduced (modified) from Chang L., Tang K., Shell M., Ringelberg C., Matteson K.J., Allman S.L. and Chen C.H., Rapid Comm. Mass Spectrom., 9, 772, 1995, with permission. 

The MALDI/TOF mass spectrum of a 25-mer oligonucleotide after enzymatic digestion with a type I phosphodiesterase at three different concentrations. The different peaks correspond to successive cleavages from the 3 side to the 5 one. Reproduced (modified) from a PerSeptive biosystems documentation, with permission. 

Negative mode FAB spectrum of an oligonucleotide with sequence UGUU. Reproduced (modified) from Grotjahn L., in Mass Spectrometry in Biomedical Research edited by Gaskell S.J., Wiley, New York, 1986, pp. 215-234, with permission. 

Collision-induced dissociation FAB/MS/MS spectrum of an oligonucleotide containing a modified base. Reproduced (modified) from Iden C.R. and Rieger R.A., Biomed. Environ. Mass Spectrom., 18, 617, 1989, with permission. 

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