Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods

Abstract

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson–Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.

mRNA Vaccines: The Dawn of a New Era of Cancer Immunotherapy

“…The i-motif is a unique DNA quadruplex structure formed by inserting two cytosine-rich duplexes into each other in an antiparallel manner only in an acidic environment. It has been reported that in an acidic microenvironment ( 50 , 51 ), nano-hydrogels can be internalized by cells to form an i-motif, which is decomposed in lysosomes ( 52 , 53 ), and the mRNA is then released into the cytoplasm to express the encoded protein. These systems include no chemical agents, so the structure is biocompatible.…”

mRNA Delivery by a pH‐Responsive DNA Nano‐Hydrogel

“…I tail and I cap are each designed to contain an i-motif sequence, which can hybridize with X tail and X cap respectively under a neutral or alkaline environment, and de-hybridize to fold into an i-motif structure under an acidic condition. [28,37] (Scheme 1c) Therefore, the nano-hydrogel is pH-responsive. It is expected to maintain the nanostructure in a physiological environment outside cells, and disintegrate to release mRNA for the expression of encoded proteins when it enters into lysosome (pH 4.5-5.0) of cells through endocytosis.…”

“…I-motif, a specific DNA quadruplex structure, is formed only in an acidic environment by two cytosine-rich double-strands being inserted into each other in antiparallel. [27,28] After being internalized by cells, with the formation of i-motif, the nano-hydrogel can disintegrate in the lysosome, which has been widely reported to have an acidic microenvironment with a pH of 4.5-5.0, [29,30] and thereby release the mRNA into the cytoplasm to express the coded protein. The architecture has excellent biocompatibility since that no chemical materials are used, and in parallel, is stable because it maintains a well-defined structure outside the cells and disintegrates only at low pH inside the cells.…”

DNA Nanodevice-Based Drug Delivery Systems

“…Photo-induced pH shift refers to the pH shift caused by the reaction of photosensitive substances under the light condition, thus transforming the structure of DNA. Zhao et al [ 98 ] fixed the photosensitive substance malachite green (MGCB) at the nanochannels of MSN, and the i-motif DNA structure was grafted onto the surface of MSN to seal the orifice; i-motif was a special DNA four-chain structure, and only when two cytosine-rich double-chains were inserted antiparallel in an acidic environment could i-motif be formed [ 99 , 100 ]. Under UV light, MGCB was dissociated into malachite green (MG) cation and OH - ion so that the pH of the solution increased, followed by the i-motif structure being converted into a single chain and the guest molecule being released [ 98 ].…”