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.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

Nanomaterials, such as metal or semiconductor nanoparticles and nanorods, exhibit similar dimensions to those of biomolecules, such as proteins (enzymes, antigens, antibodies) or DNA. The integration of nanoparticles, which exhibit unique electronic, photonic, and catalytic properties, with biomaterials, which display unique recognition, catalytic, and inhibition properties, yields novel hybrid nanobiomaterials of synergetic properties and functions. This review describes recent advances in the synthesis of biomolecule-nanoparticle/nanorod hybrid systems and the application of such assemblies in the generation of 2D and 3D ordered structures in solutions and on surfaces. Particular emphasis is directed to the use of biomolecule-nanoparticle (metallic or semiconductive) assemblies for bioanalytical applications and for the fabrication of bioelectronic devices.

Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications

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Functional Nucleic Acid Sensors

The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.

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Adding New Chemistries to the Genetic Code

The programmable and reliable hybridization of DNA strands has enabled the preparation of a wide variety of structures. This Review discusses how, in addition to these static assemblies, the process of displacing — and ultimately replacing — strands also makes possible the construction of dynamic systems such as logic gates or autonomous walkers.

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Dynamic DNA nanotechnology using strand-displacement reactions

An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of ≈109 M−1⋅min−1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.

https://www.pnas.org/content/pnas/94/9/4262.full.pdf

A General Purpose RNA-Cleaving DNA Enzyme

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.

Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coli

We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains approximately 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.

Mechanism and utility of an RNA-cleaving DNA enzyme

In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid e...

https://www.scripps.edu/barbas/pdf/Santoro00JACS.pdf

RNA cleavage by a DNA enzyme with extended chemical functionality.

With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNALys sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with l-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-l-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.

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Stephen W. Santoro

Papers

A General Purpose RNA-Cleaving DNA Enzyme

Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coli

Mechanism and utility of an RNA-cleaving DNA enzyme

RNA cleavage by a DNA enzyme with extended chemical functionality.

An expanded genetic code with a functional quadruplet codon