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This nuclease complex integrates enzymes from an extremophile single-celled organism into a nuclease with versatile nucleic acid degradation abilities. Nucleases, a class of enzymes within cells, degrade nucleic acids by breaking long strands into shorter ones. Ribonuclease (RNase) is a subclass of nucleases targeting ribonucleic acid (RNA). RNA hydrolysis is central to cell biology. The degradation of mRNA is an essential mechanism for regulating gene expression, controlled in response to environmental, development, and metabolic queues. Cells must maintain RNA quality to prevent the damaging effects of aberrant non-coding RNA accumulates or when translating defective mRNA. Since RNA participates in various cell processes, RNase is likewise essential in the background of these processes, preventing the build-up of RNA after its use and removing it when it is faulty. Different types of RNase degrade RNA from the end or center of the strand. These are known as exoribonuclease or endoribonuclease, respectively. Furthermore, RNase typically cannot degrade single-strand deoxyribonucleic acid (ssDNA) despite the structural similarity of ssDNA to RNA. A multifaceted nuclease combining endo- and exoribonuclease behavior with the ability to degrade ssDNA is a sought-after tool.
Researchers at the University of Florida have developed a nuclease complex by aggregating RecJ enzymes from the single-celled Archaeal organism, Haloferax valocanii. The end product can break down RNA from the end or center of the strand and degrade ssDNA. This broad degrading power is beneficial in cleaning processes, such as reverse transcription that leaves unwanted RNA and ssDNA.
Endo- and exonuclease functionality for degradation of RNA and ssDNA
Organisms in the domain Archaea typically live in extreme environments and, despite being prokaryotic and single-celled like most bacteria, their nucleic acids and associated enzymes function similarly to those of eukaryotes. As a result, scientists study essential biological processes such as DNA transcription and RNA degradation in archaea such as the salt-loving Haloferax volcanii. This organism contains three noteworthy nucleases: RecJ3 and RecJ4, capable of degrading ssDNA from the end, and aRNase J, an RNA degrading machine. The enzyme complex combines an aggregate of RecJ3 and RecJ4 with a variable concentration of aRNase J. This achieves on-demand tuning between endoribonuclease behavior when aRNase J is the larger portion of the ratio and exoribonuclease behavior when aRNase J is the smaller portion.
This biocatalyst, or enzyme, accelerates organic synthesis reactions in extreme conditions that available biocatalysts do not tolerate. This thermostable inorganic pyrophosphatase (PPA) developed by University of Florida researchers thermodynamically favors the hydrolysis of one mole of pyrophosphate (PPi) into two moles of orthophosphate (Pi), even in extreme conditions such as buffers with two to three moles of salt and 25% (v/v) organic solvent. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the biotechnology industry. The global market for biopharmaceuticals, $160 billion in 2011, was expected to exceed $200 billion by 2016. PPAs that are easily purified, thermostable, and solvent-tolerant are desirable for biotechnologies. However, PPAs that retain catalytic activity in high salt solutions or organic solvents are not commercially available. The haloarchaeal inorganic pyrophosphatase developed at UF is thermostable and operates in a variety of solvents, including solvents with low water activity, such as high salt solutions and organic solvents. This biocatalyst increases the solubility of hydrophobic substrates, allows for new synthetic chemistry, alters substrate specificity, eases product recovery, and reduces microbial contamination for many biotechnological applications.
Biocatalyst for biotechnological applications that is easily purified, thermostable, readily soluble, and active in conditions of low water activity.
Inorganic pyrophosphatases (PPAs) are valuable for the biotechnology industry because they catalyze the hydrolysis of pyrophosphate (PPi), a by-product of the biosynthesis of DNA, RNA, proteins, lipids, cellulose, starch, and many other compounds. The hydrolysis of PPi releases a considerable amount of energy that can push unfavorable biochemical transformations to completion. University of Florida researchers identified a novel subset of Class A Type archeal PPAs, haloarcheals, which were modeled by Haloferax volcanii PPA (HvPPA). HvPPA was then purified 357-fold by immobilized metal ion affinity chromatography and size exclusion chromatography to isolate the hexameric form of HvPPA, which is ultimately used in the hydrolysis of PPi. This haloarchaeal PPA is thermostable and beneficial for accelerating biosynthetic reactions in the forward direction in extreme conditions, such as buffers with two to three molesof salt and 25% (v/v) organic solvent.
These two novel polymerase chain reaction (PCR)-based methods enable efficient generation of substitution mutations in large plasmids (>10 kb). Site-directed mutagenesis is a common tool in the molecular biology field, allowing for a better understanding of the influence of DNA sequence on downstream biological and biochemical functions. The methods for performing site-directed mutagenesis vary but often include polymerase chain reaction (PCR) to generate point substitutions, deletions, or insertions in the DNA. The available PCR-based methods for generating point substitution mutations are typically the gold standard strategy. However, when using large plasmids, these methods present several disadvantages.
Overlap extension PCR methods involve the generation of overlapping DNA products with the desired mutations by two PCRs, subsequently used as a template for a final overlap extension. However, the final products are difficult to generate and dependent on cloning into a plasmid vector for expression, making this approach time-consuming and inefficient. An alternative and more straightforward approach incorporates complementary primer pairs with the substitution mutation at the center of each primer. Unfortunately, the formation of primer dimers is common, leading to reaction failure. Inverse PCR is another option, with one of the primers harboring the mutation near the 5’ end and the final product being phosphorylated and ligated after amplification. However, this approach is typically unreliable for large DNA templates, and DNA artifacts are frequent at the site of self-ligation. Advances are necessary for site-direct mutagenesis of large plasmids.
Researchers at the University of Florida have developed two novel PCR-based methods, “Sequential Single Primer Extension Reaction” (SSPER) and “reduce recycle PCR” (rrPCR), readily generating the desired site-directed mutations on large plasmids. These two methods are highly accurate, cost-effective, straightforward in primer design, quick, and applicable for both large and small plasmids.
PCR-based strategies for efficient, simple site-directed mutagenesis of large plasmids
These two PCR-based strategies, “Sequential Single Primer Extension Reaction” (SSPER) and “reduce recycle PCR” (rrPCR) readily generate desired site-directed modifications in large plasmids (> 10 kb). The methods incorporate easy removal of the primer(s) after the first reaction, allowing for the addition of a subsequent second reaction to generate and isolate the DNA product harboring the desired site-directed mutation(s). SSPER is a sequential design involving a single primer (p1) extension reaction followed by the removal of p1 and a similar single primer extension reaction with a second primer (p2) and the mixture of the original DNA template and newly synthesized ssDNA as the desired target.
rrPCR involves a pair of forward and reverse primers in a traditional PCR (PCR-1) style, designed to generate a PCR product of 0.1 to 1 kb with one or both primers harboring the substitution mutation in the center. Primers are not complementary, and the formation of primer dimers is minimal. After the first PCR, a simple PCR cleanup removes the primers. The resulting mixture is reduced in primers and recycled for a second PCR (PCR-2), with the dsDNA product of PCR -1 serving as the primer pair and the recycled plasmid DNA as a template again. In both cases, methylated DNA plasmids serve as templates, being removed by DpnI digestion at the end of the reactions prior to transformation into E. coli.
