Localized Molecular Automaton For In Situ Visualization Of Proteins With Specific Chemical Modifications

Chemical modification of proteins is a major means which nature uses to diversify the structure up to functionality of proteins.These modifications play important roles in regulation of the properties and activities of proteins,and govern their dynamic and intricate interactions in biological processes.Owing to the rapid development of MSbased proteomic techniques,hundreds of modifications have been identified.However,the revelation of the functions of these modifications in cellular processes is hampered by the lack of in situ imaging methods,which are capable of acquiring of spatiotemporal information of chemical modifications at protein-specific level.More importantly,proteins can be multiply modified by different groups at different residues,or at the same residue either competitively or sequentially.Thus it is of vital importance to implement the identification of both the protein and its collection of multiple modifications simultaneously,which is by far a highly challenging task.To solve the problem,we need to respectively label the target protein and modifications with probes,and design distance-dependent interactions to report the probe proximity.This work develops a localized DNA automaton,which is used to complete DNA molecular calculation on the specific protein of the cell surface.It’s capable of in situ translation of a particular protein subtype with dual chemical modifications on cell surface into a fluorescence output signal,which performs proteinconfined computation according to a designed "anticoding-coding" sequential propagation algorithm.A transmembrane glycoprotein epithelial cell adhesion molecule is chosen as the protein model.Two types of chemical modifications are introduced to cells:(1)incorporating homopropargylglycine into proteins to yield alkyne modification,which represents the direct chemical modification on amino acid residue of protein backbone;(2)tagging termini sialic acids of protein-carried glycan chains with azide groups,which illustrates an important and common protein posttranslational modification type,i.e.glycosylation.Thus the protein identity,sialic acid azidation and homopropargylglycine installation are just the three "protein characteristics" that analyzed by the automaton.The automaton contains three probes respectively for three "protein characteristics".Each probe is composed of a reaction/recognition motif as anchoring module for specific assembly of probes on "protein characteristics",and an"anticoding-coding" oligonucleotide part as signal module for playing computation role."Coding" oligonucleotides are linked with corresponding anchoring motifs.During the computation,they represent the identity of the corresponding "protein characteristics"."Anticoding" oligonucleotides originally "mask" the corresponding "coding" part and can later be triggered to propagate "identity signals" according to a predetermined calculation order between "coding" parts of probes in close spatial proximity by toehold-mediated strand displacement and restriction endonuclease nicking reactions.After anchoring the probes,once "time coding" strand is added to initiate the program,the automaton will perform three sequential YES logic computation according to "if YES then proceed" rule on individual proteins on cell surface.Only when the three types of probes are colocalized within the region of a single target protein,can the automaton complete its calculation and output a final fluorescence signal,indicating the existence of the exact subtype of protein with both modifications on cell surface.The "anticoding-coding" oligonucleotide strands and sequential propagation algorithm designed for automaton indeed form a complete set of "probe kit",which can be used for different imaging tasks:(1)To illuminate the global expression of individual"protein characteristic",one can simply use the corresponding "coding"oligonucleotide strand for each;(2)To identify proteins with single modification,the"anticoding-coding" oligonucleotide strands for the protein and the target modification need to be selected from the kit;(3)When it comes to proteins with dual modifications,the whole kit is required.This scalable feature can be attributed to the modular nature of the automata and a unique design that protein identity is set as the connecting link for checking its close proximity with dual modifications.And it can also be readily expanded to more complex algorithm by simply inserting other probe/logic layers in the cascade to reflect multi-level information of proteins.What’s more,the automaton also allows the in situ tracking of protein subtype-specific dynamic change of sialylation on cell surface in response to drugs.This work extends the use of molecular automata,opens up the possibility of using automata for in situ visualization of protein posttranslational modifications,and also paves the way for the development,screening and pharmacologic study of posttranslational modification-targeting drugs,and revelation of the sophisticated mechanisms of posttranslational modification for regulation of protein functions.