Full UV resonance Raman analysis of temperature effects on the structural arrangement of DNA segments

Abstract

Temperature dependence of vibrational spectra provides important information about the geometry and stability of nucleic acid segments. Even in cases of simple structural transitions between the folded and unfolded states, three types of spectral changes may occur – the actual structural transition, the influence of temperature change on the oligonucleotide in the folded state, and that in the unfolded state. In this work, the temperature dependences of UV resonance Raman spectroscopy (RRS) spectra excited at 244 and 257 nm were measured on two dodecadeoxynucleotides, duplex and hairpin. In both cases, the total base composition was the same, containing (in the folded state) a double‐strand section of guanine‐cytosine (GC) pairs and a central section of adenine‐thymine (AT) pairs forming a flexible part of the double helix in the duplex or the loop in the hairpin. Principal component analysis (PCA) showed that UV RRS spectrum, unlike non‐resonance RS, can reliably distinguish between the three types of temperature changes. Spectral images of the changes confirmed the B‐form double helix of GC sections in both the duplex and hairpin and different base positions in AT base sections. Additionally, minimal stacking interactions between the bases in the loop and other types of interactions of adenine with the bases of the stem in the hairpin were shown, and the main effects of heating in the folded state on the GC sections manifested weakening of stacking interactions. The results demonstrate UV RRS potential for studies of non‐canonical nucleic acid structures, providing properly analyzed and executed measurements of temperature dependence.

DNA i‐motif formation at physiological pH revealed by Raman spectroscopy

“…The 1,530 cm −1 band is found at all pHs and increases with increasing temperature, which is the same behaviour as observed for the off‐resonant Raman spectra (see Figure 2), where it is connected with thermal destabilization of the iM structure. An analogous increase in the intensity of the 1,530 cm −1 cytosine band was observed during the thermal denaturation of DNA hairpin and duplex structures 63 . This cytosine band also increases in intensity during the pH‐induced denaturation of dC 12 as documented by IR spectroscopy 64 .…”

“…Finally, the UVRRS band at ~1,650 cm −1 (both cytosine and thymine contribute to this band 62 ) tends to increase in intensity with temperature (at pH 6.6 and 8.0) for C9T3 (see Figure 4) and C9A3 (see Figure S10). Such a thermal hyperchromism was attributed to disruption of the ordered structure of DNA 63 . In addition to the bands described above, the only other bands found in the UVRRS spectra of C9T3 are around 1,060, 1,200 and 1,380 cm −1 , but these do not change with temperature.…”

“…Such a thermal hyperchromism was attributed to disruption of the ordered structure of DNA. 63 In addition to the bands described above, the only other bands found in the UVRRS spectra of C9T3 are around 1,060, 1,200 and 1,380 cm À1 , but these do not change with temperature. The main spectral contribution at $1,060 and $1,200 cm À1 likely comes from the spectrum of the glass cuvette.…”

“…An analogous increase in the intensity of the 1,530 cm À1 cytosine band was observed during the thermal denaturation of DNA hairpin and duplex structures. 63 This cytosine band also increases in intensity during the pH-induced denaturation of dC 12 as documented by IR spectroscopy. 64 Despite the temperature step of 7 C for the UVRRS melting/cooling measurements being larger compared to the step of 2 C for the off-resonant RS measurements (since each UVRRS spectrum had to be accumulated for a longer time, i.e., 30 min), some qualitative conclusions can still be drawn from the temperature-dependent UVRRS spectra.…”