Hi I have a few oligonucleotides with G4 forming sequences that I have checked they form G4 by CD spectroscopy.

I plan to double check G4 formation by using Thioflavin T (ThT) fluorescence turn‑on assay.

I have compared same G4 sequence in 3 different annealing buffers

1. 100 mM KCl, 10 mM Tris-HCl

2. 100 mM LiCl, 10 mM Tris-HCl

3. 10 mM Tris-HCl

Annealed oligonucleotide (4 uM) and ThT (2 uM) is mixed and incubated at RT for 30 mins before taking measurement at 490 nm after excitation at 425 nm.

Problem I have is that KCl buffer should give me the most stable G4 structure and therefore the highest fluorescent signal at 490 nm, but order of highest to lowest fluorescence is: LiCl > no cation > KCl

KCl is lowest...

Many published articles show K+ higher stability than Li+

My CD spectroscopy results show K+ higher stability

Is this ThT's chemical characteristic?

Any ideas or suggestions to why this could be happening would be great help

Thanks in advance