Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors.

Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism's genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro, iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms' transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods.

Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning.

The nontemplate-dependent terminal transferase activity inherent in nonproofreading DNA polymerases such as Taq provides a highly efficient method to clone PCR products. The enzyme adds a single, unpaired residue-preferentially an adenosyl residue-to each 3' end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3'-thymidyl residue at each end. The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.

Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo.

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

Quantifying DNA End Resection in Human Cells.

DNA double-strand break (DSB) end resection initiates homologous recombination (HR) and is critical for genomic stability. DSB resection has been monitored indirectly in mammalian cells using detection of protein foci or BrdU foci formation, which is dependent on single-stranded DNA (ssDNA) products of resection. Here we describe a quantitative PCR (qPCR)-based assay to directly measure levels of ssDNA intermediates generated by resection at specific DSB sites in human cells, which is more quantitative and precise with respect to the extent and efficiency of resection compared with previous methods. This assay, excluding the time for making the stable cell line expressing the restriction enzyme AsiSI fused to the estrogen receptor hormone-binding domain (ER-AsiSI), can be completed within 3 days.

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

Construction of a Novel Biosensor Based on the Self-assembly of Dual-Enzyme Cascade Amplification-Induced Copper Nanoparticles for Ultrasensitive Detection of MicroRNA153.

MicroRNAs (miRNAs) have received extensive attention because of their potential as biomarkers for cancer diagnosis and monitoring, and their effective detection is very significant. Here, a specific, one-pot, rapid, femtomolar sensitive miRNAs detection biosensor was developed based on the target-triggered three-way junction (3-WJ) and terminal deoxynucleotide transferase (TDT)/Nt.BspQI in combination with activated copper nanoparticles (CuNPs) self-assembly. To this end, a 3-WJ hairpin probe and helper probe were designed to selectively identify the target miRNA, so as to form a stable 3-WJ structure that further triggered the double-enzyme cycling to produce poly T to activate the self-assembly of CuNPs. Based on the simplicity of CuNPs generation, the poly T template fluorescence CuNPs can detect the minimum detection limit of 1 fm within 1.75 h. In addition, the applicability of this method in complex samples was demonstrated by analyzing the whole-blood RNA extraction from Parkinson patients, consisting of the results of commercial miRNA kits. The developed strategy performs powerful implications for miRNA detection, which may be beneficial for the effective diagnostic assays and biological research of Parkinson's disease.

Fluorescence determination of the activity of O6-methylguanine-DNA methyltransferase based on the activation of restriction endonuclease and the use of graphene oxide.

A fluorescence method is described for the determination of the activity of O6-methylguanine-DNA methyltransferase (MGMT). It is based on the activation of restriction endonuclease PvuII and the adsorbing a fluorophore-labelled DNA onto the surface of graphene oxide (GO). MGMT activity removes the methyl group from O6-methylguanine (O6MeG) in the fluorophore-labelled DNA to unblock the specific recognition site for further hydrolysis reaction of restriction endonuclease PvuII. The endonuclease catalytic reaction releases fluorophores (5-carboxyfluorescein) from fluorophore-labelled DNA, which can avoid fluorescence quenching by GO, creating an abundance of the fluorescence signal. The fluorescence increase in the assay is thus directly dependent on the MGMT activity. Under the optimal conditions with the emission wavelength of 519 nm (exitation at 494 nm), the activity of the MGMT can be determined in the range 0.5 to 35 ng mL-1 with a detection limit of 0.15 ng mL-1. This is extremely sensitive for the determination of MGMT. The short time of analysis (2 h) is superior to many reported strategies. The method can also be extended for the rapid and sensitive activity assay of other DNA repair enzymes by designing a proper substrate DNA. Conceivably, the technique represents a powerful tool for diagnosis and drug exploitation. Graphical abstract Schematic representation of the fluorescence method for MGMT activity assay.

DNA-mediated coupling of ATPase, translocase and nuclease activities of a Type ISP restriction-modification enzyme.

Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a ß-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.

2-Substituted 2'-deoxyinosine 5'-triphosphates as substrates for polymerase synthesis of minor-groove-modified DNA and effects on restriction endonuclease cleavage.

Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.

CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples.

Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.

Single molecule tracking reveals that the bacterial SMC complex moves slowly relative to the diffusion of the chromosome.

Structural Maintenance of Chromosomes (SMC) proteins and their complex partners (ScpA and ScpB in many bacteria) are involved in chromosome compaction and segregation in all kinds of organisms. We employed single molecule tracking (SMT), tracking of chromosomal loci, and single molecule counting in Bacillus subtilis to show that in slow growing cells, ∼30 Smc dimers move throughout the chromosome in a constrained mode, while ∼60 ScpA and ScpB molecules travel together in a complex, but independently of the nucleoid. Even an Smc truncation that lacks the ATP binding head domains still scans the chromosome, highlighting the importance of coiled coil arm domains. When forming a complex, 10-15 Smc/ScpAB complexes become essentially immobile, moving slower than chromosomal loci. Contrarily, SMC-like protein RecN, which forms assemblies at DNA double strand breaks, moves faster than chromosome sites. In the absence of Smc, chromosome sites investigated were less mobile than in wild type cells, indicating that Smc contributes to chromosome dynamics. Thus, our data show that Smc/ScpAB clusters occur at several sites on the chromosome and contribute to chromosome movement.

Single-Cell DNA Methylation Profiling: Technologies and Biological Applications.

DNA methylation is an epigenetic modification that plays an important role in gene expression regulation, development, and disease. Recent technological innovations have spurred the development of methods that enable us to study the occurrence and biology of this mark at the single-cell level. Apart from answering fundamental biological questions about heterogeneous systems or rare cell types, low-input methods also bring clinical applications within reach. Ultimately, integrating these data with other single-cell data sets will allow deciphering multiple layers of gene expression regulation within each individual cell. Here, we review the approaches that have been developed to facilitate single-cell DNA methylation profiling, their biological applications, and how these will further our understanding of the biology of DNA methylation.

Influence of uvrA, recJ and recN gene mutations on nucleoid reorganization in UV-treated Escherichia coli cells.

Exposure to ultraviolet (UV) radiation blocks DNA replication and arrests cellular division in Escherichia coli. Restoration of chromosome replication involves nucleoid reorganization, which involves the participation of the recombination-catalyzing proteins RecA, RecO, RecR and RecN. In this work, we evaluated the influence of recN, uvrA and recJ gene mutations on post-irradiation nucleoid reorganization. We used isogenic E. coli strains that are defective for these genes to study post-irradiation kinetics of the nucleoid shape fractions using fluorescence microscopy. The results showed that in the wild-type strain, post-irradiation nucleoid reorganization occurs, which restores the nucleoid shape fractions in the cells to those observed prior to irradiation. First, the nucleoid condenses into the central area of the irradiated cell. Second, the nucleoid disperses along the cell. Third, the cell enters the chromosome replicative phase and cytokinesis. Escherichia coli cells with a recN mutation did not exhibit increased nucleoid condensation, but chromosome replication and cytokinesis occurred. In the uvrA and recJ strains, the condensation step was delayed compared to the wild-type strain, and chromosome replication and cytokinesis did not occur. The results are discussed with an emphasis on the functions of RecN, UvrA and RecJ in nucleoid reorganization in UV-irradiated E. coli cells.

The H-subunit of the restriction endonuclease CglI contains a prototype DEAD-Z1 helicase-like motor.

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163-aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.

Multiplexed and Sensitive DNA Methylation Testing Using Methylation-Sensitive Restriction Enzymes "MSRE-qPCR".

DNA methylation is a chemically stable key-player in epigenetics. In the vertebrate genome the 5-methyl cytosine (5mC) has been found almost exclusively in the CpG dinucleotide context. CpG dinucleotides are enriched in CpG islands very frequently located within or close to gene promoters. Analyses of DNA methylation changes in human diagnostics have been conducted classically using methylation-sensitive restriction enzymes (MSRE). Since the discovery of bisulfite conversion-based sequencing and PCR assays, MSRE-based PCR assays have been less frequently used, although especially in the field of cancer epigenetics MSRE-based genome-wide discovery and targeted screening applications have been and are still performed successfully. Even though epigenome-wide discovery of altered DNA methylation patterns has found its way into various fields of human disease and molecular genetics research, the validation of findings upon discovery is still a bottleneck. Usually several multiples of 10 up to 100 candidate biomarkers from discovery have to be confirmed or are of interest for further work. In particular, bisulfite PCR assays are often limited in the number of candidates which can be analyzed, due to their low multiplexing capability, especially, if only small amounts of DNA are available from for example clinical specimens. In clinical research and diagnostics a similar situation arises for the analyses of cell-free DNA (cfDNA) in body fluids or circulating tumor cells (CTCs). Although tissue- or disease- (e.g., cancer) specific DNA methylation patterns can be deduced very efficiently in a genome-wide manner if around 100 ng of DNA are available, confirming these candidates and selecting target-sequences for studying methylation changes in liquid biopsies using cfDNA or CTCs remains a big challenge. Along these lines we have developed MSRE-qPCR and introduce here method details, which have been found very suitable for the efficient confirmation and testing of DNA methylation in a quantitative multiplexed manner (e.g., 48-96 plex) from ng amounts of DNA. The method is applicable in a standard qPCR setting as well for nanoliter scaled high-throughput qPCR, enabling detection of <10 copies of targets, thus suitable to pick up 0.1-1% of specific methylated DNA in an unmethylated background.

Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences.

Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.