生物化学技术

Small ribozymes such as the hairpin, hammerhead, VS, glm S, and hepatitis delta virus (HDV) are self-cleaving RNAs that are typically characterized by kinetics and structural methods. Working with these RNAs requires attention to numerous experimental details. In this chapter we focus on f ...

Hammerhead ribozymes are small catalytic RNA motifs ubiquitously present in a large variety of genomes. The reactions catalyzed by these motifs are both their self-scission and the reverse ligation reaction. Here, we describe methods for the generation of DNA templates for the subseque ...

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. We here describe a protoc ...

The development of artificial switches of gene expression is of high importance for future applications in biotechnology and synthetic biology. We have developed a powerful RNA-based system which allows for the ligand-dependent and reprogrammable control of gene expression in Es ...

A synthetic gene-regulatory device platform was described by modularly assembling three RNA components encoding distinct functions of sensing, transmission, and actuation. The molecular binding at the sensor component is translated by the transmitter component through a str ...

Molecular automata are self-operating machines serially exchanging information with their environment while changing their configurations. Molecular protoautomata are devices trained in a series of sessions with an operator to become molecular automata. Reconfigura ...

Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules that have the ability to catalyze a chemical reaction. Currently, DNAzymes have to be isolated from random-sequence DNA libraries by a process known as in vitro selection (IVS) because no naturally occurring DNAzyme has be ...

The trans insertion-splicing (TIS) reaction is a technique that can be used to site-specifically insert an RNA donor substrate into a separate RNA acceptor substrate. The TIS reaction, which is catalyzed by a group I intron-derived ribozyme from Pneumocystis carinii, is described with reg ...

Small cis-acting ribozymes have been converted into trans-acting ribozymes possessing the ability to cleave RNA substrates. The Hepatitis Delta Virus (HDV) ribozyme is one of the rare examples of these that is derived from an RNA species that is found in human cells. Consequently, it possesses ...

The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro c ...

Trans-acting hammerhead ribozymes are challenging tools for diagnostic, therapeutic, and biosensoristic purposes, owing to their specificity, efficiency, and great flexibility of use. One of the main problems in their application is related to the difficulties in the design of act ...

Hammerhead ribozymes have been extensively used as RNA-inactivating agents for therapy as well as forward genomics. A ribozyme can be designed so as to specifically pair with virtually any target RNA, and cleave the phosphodiester backbone at a specified location, thereby functionally ...

Allosteric ribozymes can be designed to respond to virtually any molecule of choice. The resulting species may be used for example as synthetic regulators of gene expression or alternatively as biosensors. In vitro selection techniques allow the isolation of active molecules from libr ...

The discovery of DNAzymes that can catalyze a wide range of reactions in the presence of metal ions is important on both fundamental and practical levels; it advances our understanding of metal–nucleic acid interactions and allows for the design of highly sensitive and selective metal ion sen ...

Nucleotide analog interference mapping (NAIM) is a quick and efficient method to define concurrently, yet singly, the importance of specific functional groups at particular nucleotide residues to the structure and function of an RNA. NAIM can be utilized on virtually any RNA with an assaya ...

Metal ions are indispensable for ribonucleic acids (RNAs) folding and activity. First they act as charge neutralization agents, allowing the RNA molecule to attain the complex active three dimensional structure. Second, metal ions are eventually directly involved in function. Nucl ...

A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ...

The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vit ...

Gene families are widely used in comparative genomics, molecular evolution, and in systematics. However, they are constructed in different manners, their data analyzed and interpreted differently, with different underlying assumptions, leading to sometimes divergent conc ...

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to r ...