Short oligonucleotides composed of fewer than 100 bases in length can form stable single stranded structures. One type of structure can occur from a strand folding back on itself and linking up with complementary bases within the sequence to form a structure in the shape of a hairpin. In fact, these structures appear naturally in several RNA and DNA fragments where the complementary bases within the strand are held together through hydrogen bonding (Watson-Crick base pairing). They are currently of interest to researchers since they are common in RNA viruses where the hairpin structure plays an important role in binding specificity. There is also interest in their use as antisense drugs since the hairpin is often resistant to enzymatic attack.
Previous studies from other researchers have uncovered several stable sequences that are capable of forming hairpins. A particular sequence containing a GAAA tetraloop is one of the shortest loops known to form stable single-stranded structure when it has complementary bases on the 5’ and 3’ sides. In the past, we have performed several kinetic and thermodynamic studies of sequences containing this tetraloop to understand how it affects hybridization. In the proposed study, we are interested in understanding how the stability in the hairpin changes when alternative nucleic acids (LNAs) are incorporated into the sequence. We will study the thermodynamics of single stranded structures using UV-Vis spectroscopy by monitoring the absorbance of the strand as a function of temperature. Since duplexed DNA has a lower extinction coefficient compared to the denatured form, at and around the melting temperature, the absorbance increases with increasing temperature signifying that duplex strands are falling apart or denaturing into two single strands. Similar melting curves can be obtained from self-hybridized strands (hairpins) and they provide information on the stability of the hairpin. We are interested in obtaining melting curves of samples where LNA is substituted into various regions of the strand. Using the melting temperatures, we can obtain information on the changes in free energy (G), enthalpy (H), and entropy (S) of the process.
Model sequences composed of GAAA tetraloops with varying numbers of complementary bases within the stem and random sequences with similar length and composition will be designed. The samples will contain DNA or a combination of DNA and LNA (these will be purchased externally). Each sample will be characterized spectrophotometrically to verify solution concentrations. Melting curves of the samples will be obtained using our existing Ocean Optics UV-Vis spectrometer with a temperature regulated cell holder. From these curves, a van’t Hoff relation will be used to obtain changes in enthalpy for the transition. Alternatively, from the melting curve, a ratio of the hairpin fraction (f) to the denatured fraction (1-f) in regions surrounding the melting temperature will be obtained and used to find the equilibrium constant (K) according to the equation below (where T is temperature and R is a constant):
By plotting ln K versus 1/T, the thermodynamic values will be determined. Tentative schedule of work Week 1~ 4 June
· Requirements for laboratory notebook
· Discuss design of oligonucleotides for study and place order
· Familiarization with equipment (pipettors, vacuum centrifuge, mixers, etc.)
I have obtained funding from SURE for the last three years. The research performed in the Summer of 2004 and 2005 on the kinetics and thermodynamics of DNA hybridization using UV-Vis spectroscopy, resulted in a publication with several students in the Journal of Biomolecular Structure and Dynamics. This research was also presented at national, regional and local meetings including a Gordon conference. It was also presented at Student Research Symposia at SUNY New Paltz. The research performed in the Summer of 2006 is being included in a manuscript for publication and will be presented at a national meeting this August.
Student Name The research will focus on the thermodynamic effects of LNA (alternative nucleic acids) substituted into different regions of the DNA strand. My goals for this study will be to master the use of the equipment and obtain data that can be applied to thermodynamic equations.
The DNA comes to us in a lyophilized (freeze dried) form from a vendor in short sequences known as oligonucleotides. These short sequences of nucleotides will contain bases that can form very stable, single stranded structures (DNA). Some strands form into a hairpin shape with one end doubled together like a paired double helix and the other into a loop (see diagram above). The main interest of the study will be to observe stabilities of the hairpin sequence when LNA’s are substituted into the strand. In order to monitor any changes I will be making solutions out of these hairpin sequences using buffers and measuring their absorbances. To take these measurements, I will be learning how to use the Ocean Optics UV-VIS spectrometer with a temperature regulated cell holder. Another way to collect data on the stability of the sequences is to measure the melting curves through self-hybridization after the LNA’s are substituted. The melting curves will be collected under various ionic strengths and buffering conditions. These values could then be used to obtain different thermodynamic quantities such as enthalpy, entropy and free energy.
This research is also important to me because I want to become a pharmacist. By working with DNA I will be able to understand it better, and be able to apply it to my pharmaceutical studies when learning about drug interactions with the human body. I feel that a research opportunity on DNA and working side by side with Dr. St. John would be a huge benefit to my current and future studies in chemistry. I understand that I will present the results of this work in the fall semester