Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Formation of Local TAT Base-Triplets in DNA Pseudoknots with Stem-Loop Complementarity
Pseudoknots belong to an interesting and diverse RNA structural motif that has significant roles in biological function; examples include: self-splicing introns, involvement in the catalytic core of various ribozymes, telomerase, riboswitches, and ribosomal frameshifting. The later one is a common mechanism found in viruses, where there is a change in the reading frame allowing for different mRNAs to be translated. This arises when a pseudoknot initiates the frameshift at the slippery sequence. The high-efficiency of frameshifting may be attributed to the formation of a local triplex within the pseudoknot, resulting in an increased interaction with the ribosome. Furthermore, the overall structure and conformation of a pseudoknot results from the actual length of the loops, number of stem base pairs and stem-loop interactions. In this work, we have used a combination of UV spectroscopy and differential scanning calorimetry (DSC) to investigate a set of DNA pseudoknots with the following sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT) where "Tn" represents a loop complementary to the stem, where n = 5, 7, 9 (PsK-9), or 11 (PsK-11). When this loop is increased higher unfolding enthalpy contributions are observed, which can be attributed to formation of one to three base-triplet stacks (15.5 kcal/mol per base-triplet stack). To confirm the formation of a local triplex in the right side of this DNA pseudoknot (see figure below), we have flipped the AT base-pair shown in red in this figure. Our DSC results show that PsK-9 and PsK-11 with one flipped AT base-pair have lower unfolding enthalpies when compared to the parent pseudoknots. To determine the actual location of the base-triplets, we have replaced adenines with 2-aminopurine (2-AP) in PsK-9 and PsK-11, as shown in the figure. We obtained fluorescence intensity changes, between the folded and unfolded states, which were much larger when 2-AP was placed near the stem-junction than at the distal end. This is in good agreement with previous findings of larger fluorescence changes when 2-AP is involved in a base triplet away from the solvent. These results confirm our finding that DNA pseudoknots can form a local triplex if the loop has the proper length to fold into the major groove with sequence complementary to the stem.
This research has been supported by Grant MCB-1122029 from NSF and GAANN grant P200A120231 from the U.S. Department of Education.
Department of Pharmaceutical Sciences