The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

/pdf/bioorthogonal-chemistry-fishing-for-selectivity-in-a-sea-of-125vhdfza6.pdf

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

/pdf/functional-nucleic-acid-sensors-k8tlieshyr.pdf

Functional Nucleic Acid Sensors

Recent advances in fluorescent probe technology have improved spatial and temporal resolution, bringing us closer to the ideal of imaging individual cellular features in real time with molecular (1–5 nm) resolution. In parallel, the development of super-resolution imaging techniques has revolutionized fluorescence microscopy. In 1873, Ernst Abbe discovered that features closer than ∼200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1–5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

/pdf/fluorescent-probes-for-super-resolution-imaging-in-living-344ty555xt.pdf

Fluorescent probes for super-resolution imaging in living cells

The review covers the knowledge on photoremovable protecting
groups and includes all relevant chromophores studied in the
time period of 2000–2012; the most relevant earlier works are
also discussed.

/pdf/photoremovable-protecting-groups-in-chemistry-and-biology-2n33qs6p7b.pdf

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

Fluorescent chemosensors for ions and neutral analytes have been widely applied in many diverse fields such as biology, physiology, pharmacology, and environmental sciences. The field of fluorescent chemosensors has been in existence for about 150 years. In this time, a large range of fluorescent chemosensors have been established for the detection of biologically and/or environmentally important species. Despite the progress made in this field, several problems and challenges still exist. This tutorial review introduces the history and provides a general overview of the development in the research of fluorescent sensors, often referred to as chemosensors. This will be achieved by highlighting some pioneering and representative works from about 40 groups in the world that have made substantial contributions to this field. The basic principles involved in the design of chemosensors for specific analytes, problems and challenges in the field as well as possible future research directions are covered. The application of chemosensors in various established and emerging biotechnologies, is very bright.

/pdf/fluorescent-chemosensors-the-past-present-and-future-d9b51ea6de.pdf

Fluorescent chemosensors: The past, present and future

The ratio of type III to type I collagen is important for properly maintaining functions of organs and cells. We propose a method to quantify the ratio of type III to total (type I + III) collagen (λIII) in a given collagen fiber bundle using second harmonic generation (SHG) light. First, the relationship between SHG light intensity and the λIII of collagen gels was examined, and the slope (k1) and SHG light intensity at 0% type III collagen (k2) were determined. Second, the SHG light intensity of a 100% type I collagen fiber bundle and its diameter (D) were measured, and the slope (k3) of the relationship was determined. The λIII in a collagen fiber bundle was estimated from these constants (k1-3) and SHG light intensity. We applied this method to collagen fiber bundles isolated from the media and adventitia of porcine thoracic aortas, and obtained λIII = 84.7% ± 13.8% and λIII = 17.5% ± 15.2%, respectively. These values concurred with those obtained with a typical quantification method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings demonstrated that the method proposed is useful to quantify the ratio of type III to total collagen in a collagen fiber bundle.

/pdf/second-harmonic-generation-light-quantifies-the-ratio-of-4g6uttsm3m.pdf

Second harmonic generation light quantifies the ratio of type III to total (I + III) collagen in a bundle of collagen fiber.

Protein-recruiting synthetic molecules targeting the Golgi surface

This review summarizes the history and most recent advances in aptamers and ribo- zymes that bind and use redox(reduction-oxidation) cofactors. Redox reactions, catalyzed by protein enzymes in an extant world, play a central role in the metabolism of numerous bio- logical molecules in the living organisms. The burden of catalyzing these reactions in a pre- protein-based world (i.e., the hypothesized RNA World) could have been borne by RNA mol- ecules. To this end, we raise a fundamental question: can RNA accelerate the redox chemical transformation? We hope that this article will be able to shed light on this intriguing ques- tion.

/pdf/ribozymes-that-use-redox-cofactors-12x48lhoeb.pdf

Ribozymes that use redox cofactors

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

/pdf/an-improved-intracellular-synthetic-lipidation-induced-59o8vinzc7.pdf

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

/pdf/a-chemogenetic-platform-for-controlling-plasma-membrane-xm6q66kelw.pdf

Shinya Tsukiji

Papers

Second harmonic generation light quantifies the ratio of type III to total (I + III) collagen in a bundle of collagen fiber.

Protein-recruiting synthetic molecules targeting the Golgi surface

Ribozymes that use redox cofactors

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation