PMC:4996378 / 20306-24200 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996378","sourcedb":"PMC","sourceid":"4996378","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996378","text":"3.2. Printing and Arraying of In Situ-Synthesized Biomolecular Ink\nThe limitations of both the leading technologies historically employed to manufacture microarrays, i.e., the synthesis-on-chip approach using photolithography and the delivery-to-chip approach using microcontact printing, have made it impractical to broaden the applicability of microarrays in the postgenomic era. Photolithography is still a very expensive and time-consuming process, and microcontact printing is restricted to a limited number of arrays and also to the deposition of presynthesized biomolecular ink. Although DNA microarrays have achieved commercial success using these approaches, it is important to note that protein microarrays have not been successful, since they are crucially dependent on maintaining the complex 3D quaternary structure of proteins on the microarray surface, which is technically difficult using these conventional approaches (Figure 1). Therefore, the concept of the on-demand in situ creation of microarrays is giving a new meaning to the term ‘custom’ microarrays, which greatly reduce the risk of loss of function when arraying biomolecular ink and also remove the need for handling steps, such as purification and modification, prior to printing. For this purpose, previously, the concept of a DNA-linked protein array was presented, where we adopted a strategy of one-to-one indexing between spatially unknown individual DNA arrays and their encoded protein array products in situ [31,32]. Next, this process was miniaturized by demonstrating the advanced application of µIP for the in situ co-synthesis and printing of protein microarrays on demand. First, magnetic beads carrying a dsDNA sequence encoding double-histidine-tagged GFP were arrayed onto a microchamber array chip. In the next step, a droplet of a cell-free-coupled transcription/translation system was sandwiched between a Ni-NTA-modified glass surface and a bead-incorporated microchamber array chip. Then, the coupled transcription/translation reaction was initiated by increasing the temperature from 4 °C to room temperature and incubating the assembly at 30 °C for 60 min. Figure 6 shows that histidine-tagged GFP was successfully synthesized in situ from the bead-bound template DNA inside the microchamber of the microchamber-array chip and transferred to the Ni-NTA-modified glass substrate to produce a clear 60-µm-scale array pattern that was successfully detected using a confocal laser microscope [22]. These results confirm the successful one-step in situ synthesis and printing of individual proteins from localized DNAs and that this approach can be used to rapidly create large-scale integrated protein microarrays directly from the encoded DNA microarrays.\nAlthough a single protein was arrayed and printed in the above demonstration, in principle, this concept is extendable to the simultaneous printing of multiple proteins per array using BEAMing and self-assembled magnetic bead approaches [23]. This has been demonstrated by fabricating kilo-giga-density DNA microarrays [24] and the on-chip synthesis and arraying of a randomized library of mutant GFP using an ultrahigh-density (144 million) microbead array format [26]. Therefore, the inclusion of an in situ synthesis process in µIP enables the development of a beyond-mega-spot protein microarray format that can be used not only to express whole proteomes on a chip, but also to create novel artificial proteins by massively increasing the repertoire of analyzable proteins up to the level used in molecular-directed evolution.\nFigure 6 Printing and arraying of in situ-synthesized protein molecules. Bright-field icroscopy image of DNA-bead carrier-incorporated microreactor array chip (left image) and fluorescence microscopy image of in situ co-synthesized and patterned GFP spots (right image). The scale bar indicates 100 µm.\n\n3","divisions":[{"label":"Title","span":{"begin":0,"end":66}},{"label":"Figure caption","span":{"begin":3588,"end":3893}}],"tracks":[]}