Selectively Identifying Exposed-over-Unexposed C–C⁺ Pairs in Human Telomeric i-Motif Structures with Length-Dependent Polymorphism

Abstract

The i-motif structure (iM) has attracted much attention, because of its in vivo bioactivity and wide in vitro applications such as DNA-based switches. Herein, the length-dependent folding of cytosine-rich repeats of the human telomeric 5′-(CCCTAA)n‑1CCC-3′ (iM-n, where n = 2–8) was fully explored. We found that iM-4, iM-5, and iM-8 mainly form the intramolecular monomer iM structures, while a tetramolecular structure populates only for iM-3. However, iM-6 and iM-7 have the potential to fold as well into the dimeric iM structures besides the monomer ones. The natural hypericin (Hyp) was used as the polymorphism-selective probe to recognize the iM structures. Interestingly, only iM-3, iM-6, and iM-7 can efficiently switch on the Hyp fluorescence by specifically binding with the outmost C–C+ base pairs that are exposed directly to solution. However, other iM structures that fold in a way with a coverage of the outmost C–C+ pairs by loop sequences are totally unavailable for the Hyp binding. Theoretical modeling indicates that adaptive π–π and cation-π interactions contribute to the Hyp recognition toward the exposed C–C+ pairs. This specific iM recognition can be boosted by a photocatalytic DNAzyme construct. Our work provides a reliable fluorescence method to selectively explore the polymorphism of iM structures.

The iMab antibody selectively binds to intramolecular and intermolecular i-motif structures

“…Although we were able to identify the best conditions for iMab to recognize its target, we still observed a clear WB band for the hTeloC 3×2 and 3×3 sequences, which, based on their sequence, would not form intramolecular iM structures. However, we reasoned that both oligonucleotides could form intermolecular iM structures (25), which would explain their binding to iMab. Multimolecular iMs have mainly been studied for their application in nanotechnology and it is generally reported that they form in sequences with short C-tracts (25, 26), and therefore show lower stability than intramolecular structures (27).…”

“…However, we reasoned that both oligonucleotides could form intermolecular iM structures (25), which would explain their binding to iMab. Multimolecular iMs have mainly been studied for their application in nanotechnology and it is generally reported that they form in sequences with short C-tracts (25, 26), and therefore show lower stability than intramolecular structures (27). To prove our hypothesis, we performed 1 H NMR analysis at two different oligonucleotide concentrations (Figure 3A), since higher DNA concentrations stimulate the formation of intermolecular structures (28).…”

“…It may therefore be advisable to use as little amount of iMab as possible for each experiment to avoid unnecessary saturation and to increase selectivity. Similarly, when titrating the target DNA concentration, it should be considered that high DNA concentrations can stimulate the formation of intermolecular quadruplex structures (25, 26). We have shown by NMR that the hTeloC variants used by Boissieras et al and in this study are capable of forming intermolecular iMs (Figure 3A-B), providing a direct explanation for the apparent discrepancies reported by the authors, and confirming the specificity of the iMab antibody.…”