Nucleic acid amplification is the basis for many molecular diagnostic assays. In these cases, the amplification product must be detected and analyzed, typically requiring extended workflow time, sophisticated equipment, or both. Here we present a novel method of amplification detection that harnesses the pH change resulting from amplification reactions performed with minimal buffering capacity. In loop-mediated isothermal amplification (LAMP) reactions, we achieved rapid (<30 min) and sensitive (<10 copies) visual detection using pH-sensitive dyes. Additionally, the detection can be performed in real time, enabling high-throughput or quantitative applications. We also demonstrate this visual detection for another isothermal amplification method (strand-displacement amplification), PCR, and reverse transcription LAMP (RT-LAMP) detection of RNA. The colorimetric detection of amplification presented here represents a generally applicable approach for visual detection of nucleic acid amplification, enabling molecular diagnostic tests to be analyzed immediately without the need for specialized and expensive instrumentation.

Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from H a e n q~h i l u s parainfluenzae ( H p a l ) , were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and K,,, for ligated and renatured RNase A were 8.2 s -' and 1.5 mM, in good agreement with reported values of 8.3 s -' and 1.2 mM (Hodges & Merrifield, 1975). Ligated Hpal had a specific activity of 0.5-1.5 X 10' U/mg, which compared favorably with the expected value of 1-2 X 10' U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.

SUMMARY Like membranous organelles, large-scale coassembly of macromolecules can organize functions in cells. Ribonucleoproteins (RNPs) can form liquid or solid aggregates, but control and consequences of these RNP states in living, developing tissue are poorly understood. Here, we show that regulated RNP factor interactions drive transitions among diffuse, semiliquid, or solid states to modulate RNP sorting and exchange in the Caenorhabditis elegans oocyte cytoplasm. Translation repressors induce an intrinsic capacity of RNP components to coassemble into either large semiliquids or solid lattices, whereas a conserved RNA helicase prevents polymerization into nondynamic solids. Developmental cues dramatically alter both fluidity and sorting within large RNP assemblies, inducing a transition from RNP segregation in quiescent oocytes to dynamic exchange in the early embryo. Therefore, large-scale organization of gene expression extends to the cytoplasm, where regulation of supramolecular states imparts specific patterns of RNP dynamics.

No abstract

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E(C)) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E(C), even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.

Mutations in somatic cells generate a heterogeneous genomic population and may result in serious medical conditions. Although cancer is typically associated with somatic variations, advances in DNA sequencing indicate that cell-specific variants affect a number of phenotypes and pathologies. Here, we show that mutagenic damage accounts for the majority of the erroneous identification of variants with low to moderate (1 to 5%) frequency. More important, we found signatures of damage in most sequencing data sets in widely used resources, including the 1000 Genomes Project and The Cancer Genome Atlas, establishing damage as a pervasive cause of sequencing errors. The extent of this damage directly confounds the determination of somatic variants in these data sets.

The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system. Furthermore, the M. xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.

No abstract