Poster abstracts

Poster number 78 submitted by Sharla Wood

Bulk and single molecule analysis of the effects of 2’ modifications on molecular beacons

Sharla Wood (Chemistry, Wayne State University), David Rueda (Chemistry, Wayne State University)

Abstract:
In this study, we have used fluorescence resonance energy transfer (FRET) and single molecule spectroscopy to analyze the effects that 2’ backbone modifications have on the dynamics of molecular beacons (MB). Molecular beacons are advantageous for live cell assays because they only fluoresce in the presence of the target molecule, negating the need to wash away unbound probes, which allows delivery into living cells. Molecular beacons with 2’ backbone modifications are expected to prevent nuclease degradation in the cells. 2’F, 2’OMe, and 2’H modifications were characterized and compared to the parent 2’OH MB via steady state, time resolved, and single molecule FRET under physiological conditions. These studies indicate that the modified MBs act similarly to the 2’OH MB. The investigated MBs bind with similar affinity to the RNA target. The 2’OMe MB binds with 3-fold better affinity than the 2’OH MB, while the 2’F binds with similar affinity and 2’H MBs binds with 3-fold less. Single molecule studies show a lack of MB dynamics observed in the minute timescale, as well as a large dynamic range in that the molecular beacon alone was primarily in the closed conformation and, in the presence of the target, the beacon was primarily in the open conformation.

Keywords: molecular beacon, single molecule FRET