Effects of secondary structure stability on oligonucleotide electrophoretic mobility

Christiaan A Meadows, Purdue University

Abstract

It is now known that certain nucleic acid sequences assume secondary structures, such as hairpin loops, which are unusually stable. DNA molecules which contain such structural elements often exhibit aberrant mobility patterns during electrophoretic separation. The stability of secondary structural elements in nucleic acid molecules and their implications for both in-vitro and in-vivo applications are thus becoming a subject of growing interest. Presently, a small but increasing number of ultra stable DNA hairpin molecules have been studied. It is unlikely, however, that all such sequences have been identified, that the factors contributing to their stability are well understood, and that their implications for biological and laboratory processes have been fully elucidated. After demonstrating the dependence of the electrophoretic mobility of short deoxyribonucleotide hairpins on the stability of the structures which they assume, the relationship between the two has been exploited to allow selection and study of unusually stable hairpin molecules from DNA oligonculeotide libraries, utilizing polyacrylamide gel electrophoresis (PAGE). The correlation between electrophoretic mobility and thermodynamic stability, was established by demonstrating that: (1) there was a significant and reproducible mobility difference between comparable molecules in the hairpin and random coil conformations, (2) that hairpins containing differing loop sequences have different mobility enhancements, (3) that these mobility enhancements were reduced by treatments known to disrupt secondary structure, and (4) that molecules with the highest Tm and α values also manifest the greatest relative mobility enhancements. A system for selecting and sequencing ultra stable hairpins was then developed. The efficacy of this selection system was demonstrated by its ability to isolate sequences known to form ultra stable structures GACGGAGTC, GACGAAGTC, GACGTAGTC, and GACGCAGTC from the GACNNNGTC oligonucleotide library. The sequences of the selected molecules were verified via matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy. The contribution of each base in the loop region to the overall stability of the GACGCAGTC tri-loop hairpin and the unusually stable CG AGTTTGACG heptaloop was probed via analysis of the PAGE separation pattern of N-substituted mini-libraries and by the subsequent MALDI-TOF sequencing of the band in each separation lane.

Degree

Ph.D.

Advisors

Weith, Purdue University.

Subject Area

Biochemistry

COinS