Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

Abstract

DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a 'handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the 'handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.

A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

“…We have realized nucleic-acid-based nanodevices that precisely report the pH associated with endosomal maturation 16,20 and address their crosstalk with the secretory pathway 17 . These molecular devices can be coupled to simple organelle localization strategies that co-opt specific cellular trafficking pathways 17,21 . Here, we integrate these trafficking strategies to specifically localize a chloride-sensitive DNA-based nanodevice-called Clensorwithin organelles along the endolysosomal pathway.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…As such, the i‐motif can be considered to be an aptamer for proton sensing. The pH ranges sensed by the i‐motif can be shifted toward more acidic or basic pH through the incorporation of modified bases or different numbers of cytosine bases . The I‐switch consisted of two DNA duplexes that were linked together through a flexible tether and contained fluorophore‐labeled i‐motif forming overhangs at both ends (Figure A).…”

“…Moreover, the modular nature of the I‐switch allows custom functionalization with proteins as well as with different FRET pairs. This strategy was later extended for the detection of pH changes associated with the trans ‐Golgi and cis ‐Golgi networks, and the endoplasmic reticulum …”

Nucleic Acid–Based Nanodevices in Biological Imaging

“…pH reporters based on the i-motif illustrate how one can tune reporter characteristics of the measuring module. By increasing the number of cytosines, one can shift the K D to higher pH (115). However, this shift increases the cooperativity of the pH-induced structural transition and simultaneously narrows the overall pH regime that the device reports on.…”