Kinetics of End-to-End Collision in Short Single-Stranded Nucleic Acids

A novel fluorescence-based method, which entails contact quenching of the long-lived fluorescent
state of 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO), was employed to measure the kinetics of end-to-end
collision in short single-stranded oligodeoxyribonucleotides of the type 5‘-DBO−(X)n-dG with X = dA, dC,
dT, or dU and n = 2 or 4. The fluorophore was covalently attached to the 5‘ end and dG was introduced
as an efficient intrinsic quencher at the 3‘ terminus. The end-to-end collision rates, which can be directly
related to the efficiency of intramolecular fluorescence quenching, ranged from 0.1 to 9.0 × 106 s-1. They
were strongly dependent on the strand length, the base sequence, as well as the temperature.
Oligonucleotides containing dA in the backbone displayed much slower collision rates and significantly
higher positive activation energies than strands composed of pyrimidine bases, suggesting a higher intrinsic
rigidity of oligoadenylate. Comparison of the measured collision rates in short single-stranded oligodeoxyribonucleotides with the previously reported kinetics of hairpin formation indicates that the intramolecular
collision is significantly faster than the nucleation step of hairpin closing. This is consistent with the
configurational diffusion model suggested by Ansari et al. (Ansari, A.; Kuznetsov, S. V.; Shen, Y. Proc.
Natl. Acad. Sci. USA 2001, 98, 7771−7776), in which the formation of misfolded loops is thought to slow
hairpin formation.