Chemical messengers are small, diffusible molecules within living organisms and include second messengers such as ions, hormones, neurotransmitters, cyclic nucleotides etc that play a central role in development and cell function. Methods that are generalized to capture maps of chemical messengers within cells are invaluable in order to understand the manifold functions of second messengers. Methods to obtain chemical maps specific to a given messenger mostly use genetically encodable fluorescent sensors.
In contrast, methods that are generalizable to multiple second messengers tend to use imaging based on CARS, SIMS and MALDI, still need to achieve either adequate temporal resolution or reduce imaging artifacts. Thus obtaining high resolution spatiotemporal chemical maps in living systems using a generalizable methodology is still challenging. A second messenger that plays a crucial role in metabolism, neuronal activity, cell-cycle control and growth is pH. Different second messengers are functionally coupled and act in concert to stringently maintain sub-cellular proton concentrations since pH regulates the activity of key enzymes and ion channels.
Subcellular organelles are bounded by membranes where the intra-organellar pH is stringently regulated. The lumenal pH of various cellular organelles has been mapped primarily using pH-sensitive fluorophore functionalised ligands that bind specifically to receptors that are resident in the relevant organelles. As the receptor shuttles between the plasma membrane and its relevant organelles, the derivatised ligand is ferried by the receptor along its retrograde endocytic pathway. Organelle pH may also be measured by expressing pH sensitive fluorescent proteins fused to peptide sequences that function as organelle localization signals. However these cannot give temporal information on pH changes of a receptor containing compartment while it undergoes maturation.
Thus, most studies have utilized pH reporters in the form of fluorescent pH probes conjugated to endocytosable ligands. However, for the vast majority of cases where (a) the fluorophore conjugation to the ligand either disrupts ligand structure or ligand trafficking and (b) for membrane proteins which have no associated ligands, pH mapping of the relevant compartmental maturation process is still not accessible.
The burgeoning field of DNA nanotechnology has yielded a number of powerful synthetic molecular devices for small molecule sensing in vitro. Yet remarkably, this chemical diversity in sensing has not yet been exploited in cellulo or in vivo. One of the rare examples of DNA-based molecular devices that show quantitative preservation of its sensing functionality both in cellulo and in vivo, is the I-switch. This is a DNA assembly that undergoes a conformational change triggered by acidic pH. Acidic pH causes the formation of a non-Watson-Crick based DNA motif called the I-tetraplex, or i-motif that is then transduced into a large scale conformational change of the overall DNA assembly. The I-switch has therefore been used in living systems as a pH sensor to map spatial and temporal pH changes associated with the maturation of endosomes, by conjugating it with endocytic ligands. However, such DNA devices are not amenable to report on the chemical environments of the vast majority of proteins.
DNA has been used to build nanomechanical devices with potential in cellulo and in vivo applications. However, their in cellulo applications in different biological pathways are limited due to current device response times as well as limitations associated with their delivery to precise intracellular locations.