A DNA walker based on hairpin-shaped DNA aligner and fueled by nicking endonuclease for sensitive and rapid miRNA analysis.

BACKGROUND: DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy. RESULTS: A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums. SIGNIFICANCE: The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.

A novel Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 possesses 5'-3' exonuclease and endonuclease activities.

Mre11 is one of important proteins that are involved in DNA repair and recombination by processing DNA ends to produce 3'-single stranded DNA, thus providing a platform for other DNA repair and recombination proteins. In this work, we characterized the Mre11 protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-Mre11) biochemically and dissected the roles of its four conserved residues, which is the first report on Mre11 proteins from Thermococcus. Tba-Mre11 possesses exonuclease activity for degrading ssDNA and dsDNA in the 5'-3' direction, which contrasts with other reported Mre11 homologs. Maximum degradation efficiency was observed with Mn2+ at 80 °C and at pH 7.5-9.5. In addition to possessing 5'-3' exonuclease activity, Tba-Mre11 has endonuclease activity that nicks plasmid DNA and circular ssDNA. Mutational data show that residues D10, D51 and N86 in Tba-Mre11 are essential for DNA degradation since almost no activity was observed for the D10A, D51A and N86A mutants. By comparison, residue D44 in Tba-Mre11 is not responsible for DNA degradation since the D44A mutant possessed the similar WT protein activity. Notably, the D44A mutant almost completely abolished the ability to bind DNA, suggesting that residue D44 is essential for binding DNA.

The PR-10 Protein Pru p 1 is an Endonuclease that Preferentially Cleaves Single-Stranded RNA.

Pathogenesis-related class 10 (PR-10) proteins play a crucial role in plant defense by acting as ribonucleases. The specific mechanism of action and substrate specificity of these proteins have remained largely unexplored so far. In this study, we elucidate the enzymatic activity of Pru p 1, a PR-10 protein from peach. We demonstrate that this protein catalyzes the endonucleolytic backbone cleavage of RNA substrates into short oligonucleotides. Initial cleavage products, identified through kinetic analysis, can bind again, priming them for further degradation. NMR binding site mapping reveals that the large internal cavity of Pru p 1, which is characteristic for PR-10 proteins, serves as an anchoring site for single-stranded ribonucleotide chains. We propose a structure-based mechanistic model that accounts for the observed cleavage patterns and the inhibitory effect of zeatin, a nucleoside analog, on the ribonuclease activity of Pru p 1.

Biophysical and structural study of La Crosse virus endonuclease inhibition for the development of new antiviral options.

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors.

In vitro detection of circulating tumor cells using the nicking endonuclease-assisted lanthanide metal luminescence amplification strategy.

The in vitro detection of circulating tumor cells (CTCs) has been proven as a vital method for early diagnosis and evaluation of cancer metastasis, since the existence and number fluctuation of CTCs have shown close correlation with clinical outcomes. However, it remains difficult and technically challenging to realize accurate CTCs detection, due to the rarity of CTCs in the blood samples with complex components. Herein, we reported a CTCs in vitro detection strategy, utilizing a loop amplification strategy based on DNA tetrahedron and nicking endonuclease reaction, as well as the anti-background interference based on lanthanide metal luminescence strategy. In this work, a detection system (ATDN-MLLPs) composed of an aptamer-functionalized tetrahedral DNA nanostructure (ATDN) and magnetic lanthanide luminescent particles (MLLPs) was developed. ATDN targeted the tumor cells via aptamer-antigen recognition and extended three hybridizable target DNA segments from the apex of a DNA tetrahedron to pair with probe DNA on MLLPs. Then, the nicking endonuclease (Nt.BbvCI) recognized the formed double-strand DNA and nicked the probe DNA to release the target DNA for recycling, and the released TbNps served as a high signal-to-noise ratio fluorescence signal source for CTCs detection. With a detection limit of 5 cells/mL, CTCs were selectively screened throughout a linear response range of low orders of magnitude. In addition, the ATDN-MLLPs system was attempted to detect possible existence of CTCs in biological samples in vitro.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Biochemical characterization and mutational analysis of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Archaeal NurA protein plays a key role in producing 3'-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5'-3' exonuclease activity for degrading DNA, displaying maximum efficiency at 45 °C-65 °C and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.

Activation of CBASS Cap5 endonuclease immune effector by cyclic nucleotides.

The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS-STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae (Ps) with second messengers. The key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.

Template and target-site recognition by human LINE-1 in retrotransposition.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.