Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427–1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.

The photostability of cyanine-styryl dyes of the indole-quinolinium type can be significantly improved by structural variations while the excellent optical properties including the bright fluorescence in the presence of DNA can be maintained or even improved, too.

A new cyano‐substituted thiazole red derivative as a red emitter and a novel green fluorescent donor dye of the cyanine styryl type were synthesized in good yields. Characterization of their optical properties revealed excellent photostabilities and large apparent Stokes' shifts. Both dyes can be incorporated into oligonucleotides through postsynthetic “click”‐type chemistry and combined in a diagonal arrangement in double‐stranded DNA. As a result, both dyes combine to an energy‐transfer pair in DNA that shows remarkable optical properties such as significant emission wavelength shift from green to red upon hybridization owing to high energy‐transfer efficiency between the two dyes and remarkable emission red/green color contrast ratio. The combination of these dyes as an energy‐transfer pair according to our concept of “DNA traffic lights” has high potential for applications in molecular imaging.

Fluorescent bioanalytics and fluorescent cell imaging of nucleic acids demands the design and synthesis of new dyes that exhibit potentially interesting optical properties particularly in combination with other dyes.[1] Dual covalent labeling of nucleic acids has turned out to be very useful for creating tailor-made fluorescent probes for such applications. [2] For instance, hybridization-sensitive probes can be created by two covalently attached thiazole orange (TO) fluorophores that interact excitonically only in the single strand. [3] On the other hand, observable fluorescence color changes can be achieved by applying an energy transfer that occurs between two adjacent fluorescent labels that are forced into close proximity by the surrounding DNA architecture. [4] With respect to the preparation of such dual labeled oligonucleotides recent developments of the "click"-type Huisgen-Sharpless-Meldal cycloaddition for nucleic acids offers great advantages since (unexpectedly) complicated and time-consuming syntheses of DNA building blocks for potentially interesting dyes can be avoided.[5]The cyanine-styryl-type dyes represent very promising candidates for nucleic acids based primarily on their recently reported property as bright noncovalent binders to RNA.[6] Herein, we present the synthesis of a "clickable" CyIQ (cyanine indole quinoline) fluorophore that can be easily attached covalently to any desired 2'-position in nucleic acids. Moreover, the optical properties of the CyIQ dye can be tuned by excitonic interaction with a second adjacent label, or by energy transfer processes with thiazole red (TR) [7] as a second fluorescence base surrogate. The CyIQ dye consists of a quinoline part that is conjugated with an indole by a styryl-type bridge. The dye can be prepared in a four-step synthesis including the introduction of the "clickable" azide functionality (Scheme 1). It starts with commercially available 2-methyl quinoline (1) that is alkylated with 3-iodopropanole (2) as the short linker between the dye and the azide group. Condensation of the methyl group of 3 in the presence of piperidine yields dye 5. In the next steps, an Appel reaction leads to iodide 6, and the azide is introduced by nucleophilic substitution to yield 7, ready for postsynthesis DNA modification.Due to its positive charge the CyIQ dye 5 is sufficiently water soluble for titration experiments with a random-sequence double-stranded DNA (Figure 1). The absorption spectra of this titration reveal significant changes; especially the absorption shift from 465 nm (without DNA) to 472 nm (with DNA) indicates excitonic dye-dye interactions of 5 (in the absence of DNA), which are interrupted by the interaction of the dye with an increasing amount of DNA. These dye-dye interactions cause a significant fluorescence quenching of 5 in aqueous solution without DNA. With increasing amounts of DNA the fluorescence intensity of the dye is recovered and finally enhanced to the 17-fold when the saturation plateau is reached. This fluorescence enhancemen...