Nuclease

About: Nuclease is a research topic. Over the lifetime, 5497 publications have been published within this topic receiving 204897 citations.

An efficient mRNA-dependent translation system from reticulocyte lysates.

Abstract: A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N′-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

Oligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation

Abstract: We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.

Endonuclease G is an apoptotic DNase when released from mitochondria

Abstract: Nucleosomal fragmentation of DNA is a hallmark of apoptosis (programmed cell death), and results from the activation of nucleases in cells undergoing apoptosis. One such nuclease, DNA fragmentation factor (DFF, a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD)), is capable of inducing DNA fragmentation and chromatin condensation after cleavage by caspase-3 (refs 2,3,4). However, although transgenic mice lacking DFF45 or its caspase cleavage site have significantly reduced DNA fragmentation, these mice still show residual DNA fragmentation and are phenotypically normal. Here we report the identification and characterization of another nuclease that is specifically activated by apoptotic stimuli and is able to induce nucleosomal fragmentation of DNA in fibroblast cells from embryonic mice lacking DFF. This nuclease is endonuclease G (endoG), a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis. Once released from mitochondria, endoG cleaves chromatin DNA into nucleosomal fragments independently of caspases. Therefore, endoG represents a caspase-independent apoptotic pathway initiated from the mitochondria.

Crystal structure of Cas9 in complex with guide RNA and target DNA

Abstract: The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Allelic discrimination using fluorogenic probes and the 5' nuclease assay.

Abstract: Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5' nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5' nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.