Biochemical Compounds

Biochemical Compounds

ISSN 2052-9341
Original Research

Melting behavior of pseudoknots containing adjacent GC and AT rich domains

Calliste Reiling-Steffensmeier, Mikenzie Nordeen and Luis A. Marky*

*Correspondence: Luis A. Marky lmarky@unmc.edu

Author Affiliations

Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, United States.

Abstract

Background: Z-DNA is very important in transcription when RNA polymerase transcribes and Z-DNA is formed on the 5’ end following the polymerase. Pseudoknots have also been found to play important roles in the biology of RNA, such as the expression of genes and in the overall tertiary structure of RNA molecules.

Methods: A combination of UV and circular dichroism (CD) spectroscopies and differential scanning calorimetry (DSC) were used to investigate the UV/CD spectral characteristics and the unfolding thermodynamics of two DNA pseudoknots with the following sequences: d(CGCGCGT5GAAATTCCGCGCGT5 GAATTTC) (CG-PsK) and d(GCGCGCT5CAAATT-GGCGCGCT5CAATTTG) (GC-PsK), where “T5” are loops of five thymines., along with, two control self-complementary duplexes, d(CG)3 (Du-CG) and d(GC)3 (Du-GC).

Results: The UV/DSC melts showed sequential biphasic (CG-PsK) and triphasic (GC-PsK) transitions, with TMs independent of the strand concentration. Their DSC unfolding took place in two independent parts, the unfolding of the AT rich stem followed by the GC rich stem. CG-PsK’s thermodynamic profiles under both salt conditions were similar, while the stability and the folding enthalpy were lower in this salt range for GC-PsK. The CD spectrum showed the d(CG)3 portion of the stem of CG-PsK flips into a left handed helix in high salt conditions that could be affecting the loop/stem interplay, yielding a higher folding enthalpy relative to GC-PsK.

Conclusions: Each pseudoknot formed intramolecularly with sequential transitions corresponding to the
associated AT and GC rich melting domains. The placement of d(CG)3 in CG-PsK yielded a CD spectra with a partial transition of the full stem to a left-handed helix. The melting behavior of CG-PsK remained unchanged with the addition of salt, while GC-PsK was greatly affected due to a more constrained right stem of the pseudoknot. The main reasons for these differences was the melting behavior of the AT rich motif, which is explained in terms of a loop/stem interplay within GC-PsK, hydration differences of their dA•dT base pairs, and/or the conversion of CG-PsK to a partial left handed helix. This conversion could be helping to stabilize this pseudoknot allowing the right stem to be less constrained at 5 M NaCl.

Keywords: Intramolecular DNA structures, pseudoknots, thermodynamics, differential scanning calorimetry, circular dichroism, left-handed DNA

ISSN 2052-9341
Volume 5
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