Efficient manipulation of gene expression using Natronobacterium gregoryi Argonaute in zebrafish.

BACKGROUND: Natronobacterium gregoryi Argonaute (NgAgo) was found to reduce mRNA without generating detectable DNA double-strand breaks in a couple of endogenous genes in zebrafish, suggesting its potential as a tool for gene knockdown. However, little is known about how it interacts with nucleic acid molecules to interfere with gene expression. RESULTS: In this study, we first confirmed that coinjection of NgAgo and gDNA downregulated target genes, generated gene-specific phenotypes and verified some factors (including 5' phosphorylation, GC ratio, and target positions) of gDNAs affecting gene downregulation. Therein, the sense and antisense gDNAs were equally effective, suggesting that NgAgo possibly binds to DNA. NgAgo-VP64 with gDNAs targeting promoters upregulated the target genes, further providing evidence that NgAgo interacts with genomic DNA and controls gene transcription. Finally, we explain the downregulation of NgAgo/gDNA target genes by interference with the process of gene transcription, which differs from that of morpholino oligonucleotides. CONCLUSIONS: The present study provides conclusions that NgAgo may target genomic DNA and that target positions and the gDNA GC ratio influence its regulation efficiency.

NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

The prokaryotic Argonaute proteins enhance homology sequence-directed recombination in bacteria.

Argonaute proteins are present and conserved in all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participates in host defense by DNA interference. Here, we report that the Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertions or deletions in Pasteurella multocida and Escherichia coli at efficiencies of 80-100%. Additionally, the effects are in a homologous arms-dependent but guide DNA- and potential enzyme activity-independent manner. Interestingly, such effects were also observed in other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo). The underlying mechanism of the NgAgo system is a positive selection process mainly through its PIWI-like domain interacting with recombinase A (recA) to enhance recA-mediated DNA strand exchange. Our study reveals a novel system for enhancing homologous sequence-guided gene editing in bacteria.

Structural Evolution of a Retinal Chromophore in the Photocycle of Halorhodopsin from Natronobacterium pharaonis.

We revealed the chloride ion pumping mechanism in halorhodopsin from Natronobacterium pharaonis ( pHR) by exploring sequential structural changes in the retinal chromophore during its photocycle using time-resolved resonance Raman (RR) spectroscopy on the nanosecond to millisecond time scales. A series of RR spectra of the retinal chromophore in the unphotolyzed state and of the three intermediates of pHR were obtained. Using singular value decomposition analysis of the C═C and C-C stretch bands in the time-resolved RR spectra, we identified the spectra of the K, L, and N intermediates. We focused on structural markers of the RR bands to explore the structure of the retinal chromophore. In the unphotolyzed state, the retinal chromophore is in the planar all- trans, 15- anti geometry. The bound ion affects the polyene chain but does not interact with the protonated Schiff base. In the observed intermediates, the chromophore is in the 13- cis configuration. The chromophore in the K intermediate is distorted due to the photoisomerization of retinal. The hydrogen bond is weak in the unphotolyzed state and in the K intermediate, resulting in exchange of the hydrogen-bond acceptor to a water molecule in the K-to-L transition, relaxation of the polyene chain distortion, and generation of an alternative distortion near the Schiff base. The bound halide ion interacts with the protonated Schiff base through the water molecule bound to the protonated Schiff base. In the L-to-N transition, the hydrogen acceptor of the protonated Schiff base switches from the water molecule to another species, although the strong hydrogen bond of the protonated Schiff base remains. This paper reports the first observation of sequential changes in the RR spectra in the pHR photocycle, provides information on the structural evolution of the retinal chromophore, and proposes a model for chloride ion translocation in pHR.

The therapeutic landscape of HIV-1 via genome editing.

Current treatment for HIV-1 largely relies on chemotherapy through the administration of antiretroviral drugs. While the search for anti-HIV-1 vaccine remain elusive, the use of highly active antiretroviral therapies (HAART) have been far-reaching and has changed HIV-1 into a manageable chronic infection. There is compelling evidence, including several side-effects of ARTs, suggesting that eradication of HIV-1 cannot depend solely on antiretrovirals. Gene therapy, an expanding treatment strategy, using RNA interference (RNAi) and programmable nucleases such as meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR-Cas9) are transforming the therapeutic landscape of HIV-1. TALENS and ZFNS are structurally similar modular systems, which consist of a FokI endonuclease fused to custom-designed effector proteins but have been largely limited, particularly ZFNs, due to their complexity and cost of protein engineering. However, the newly developed CRISPR-Cas9 system, consists of a single guide RNA (sgRNA), which directs a Cas9 endonuclease to complementary target sites, and serves as a superior alternative to the previous protein-based systems. The techniques have been successfully applied to the development of better HIV-1 models, generation of protective mutations in endogenous/host cells, disruption of HIV-1 genomes and even reactivating latent viruses for better detection and clearance by host immune response. Here, we focus on gene editing-based HIV-1 treatment and research in addition to providing  perspectives for refining these techniques.

Cation binding to halorhodopsin.

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl(-) pump. It was recently demonstrated that the Natronomonas pharaonis halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn(2+) and Ca(2+) bind to the protein. Halorhodopsin has a high affinity for Mn(2+) ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn(2+) ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn(2+) ions, as well as Cl(-) ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl(-) anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration.

Integral membrane protein structure determination using pseudocontact shifts.

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

Long-term monitoring of ultratrace nucleic acids using tetrahedral nanostructure-based NgAgo on wearable microneedles.

Real-time and continuous monitoring of nucleic acid biomarkers with wearable devices holds potential for personal health management, especially in the context of pandemic surveillance or intensive care unit disease. However, achieving high sensitivity and long-term stability remains challenging. Here, we report a tetrahedral nanostructure-based Natronobacterium gregoryi Argonaute (NgAgo) for long-term stable monitoring of ultratrace unamplified nucleic acids (cell-free DNAs and RNAs) in vivo for sepsis on wearable device. This integrated wireless wearable consists of a flexible circuit board, a microneedle biosensor, and a stretchable epidermis patch with enrichment capability. We comprehensively investigate the recognition mechanism of nucleic acids by NgAgo/guide DNA and signal transformation within the Debye distance. In vivo experiments demonstrate the suitability for real-time monitoring of cell-free DNA and RNA with a sensitivity of 0.3 fM up to 14 days. These results provide a strategy for highly sensitive molecular recognition in vivo and for on-body detection of nucleic acid.

Large deformation of helix F during the photoreaction cycle of Pharaonis halorhodopsin in complex with azide.

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.

Natronobacterium texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of a former lake.

A novel haloalkaliphilic archaeon, strain B23(T) was isolated from the former lake Texcoco in Mexico. The strain was Gram-stain-negative, the cells coccoid to ovoid rods, red pigmented and aerobic. Strain B23(T) grew in 1.7-4.3 M NaCl, at pH 6.5-9.5 and at 25-45 °C with optimal growth at 2.6-3.4 M NaCl, pH 7.5-8.5 and 37 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B23(T) was most closely related to Natronobacterium gregoryi SP2(T) with 97.3 % sequence similarity. The polar lipids of strain B23(T) were phosphatidylglycerol and several unidentified phospholipids. The G+C content of the DNA of the strain was 62.5 mol%. Levels of DNA-DNA relatedness between strain B23(T) and Natronobacterium gregoryi DSM 3393(T) was 32.3 %. The name Natronobacterium texcoconense sp. nov. is proposed. The type strain is B23(T) ( = CECT 8068(T) = JCM 17655(T)).

Comparative simulations of the ground state and the M-intermediate state of the sensory rhodopsin II-transducer complex with a HAMP domain model.

The complex of sensory rhodopsin II (SRII) and its cognate transducer HtrII (2:2 SRII-HtrII complex) consists of a photoreceptor and its signal transducer, respectively, associated with negative phototaxis in extreme halophiles. In this study to investigate how photoexcitation in SRII affects the structures of the complex, we conducted two series of molecular dynamics simulations of the complex of SRII and truncated HtrII (residues 1-136) of Natronomonas pharaonis linked with a modeled HAMP domain in the lipid bilayer using the two crystal structures of the ground state and the M-intermediate state as the starting structures. The simulation results showed significant enhancements of the structural differences observed between the two crystal structures. Helix F of SRII showed an outward motion, and the C-terminal end of transmembrane domain 2 (TM2) in HtrII rotated by ∼10°. The most significant structural changes were observed in the overall orientations of the two SRII molecules, closed in the ground state and open in the M-state. This change was attributed to substantial differences in the structure of the four-helix bundle of the HtrII dimer causing the apparent rotation of TM2. These simulation results established the structural basis for the various experimental observations explaining the structural differences between the ground state and the M-intermediate state.

Color Tuning in rhodopsins: the origin of the spectral shift between the chloride-bound and anion-free forms of halorhodopsin.

Detailed knowledge of the molecular mechanisms that control the spectral properties in the rhodopsin protein family is important for understanding the functions of these photoreceptors and for the rational design of artificial photosensitive proteins. Here we used a high-level ab initio QM/MM method to investigate the mechanism of spectral tuning in the chloride-bound and anion-free forms of halorhodopsin from Natronobacterium pharaonis (phR) and the interprotein spectral shift between them. We demonstrate that the chloride ion tunes the spectral properties of phR via two distinct mechanisms: (i) electrostatic interaction with the chromophore, which results in a 95 nm difference between the absorption maxima of the two forms, and (ii) induction of a structural reorganization in the protein, which changes the positions of charged and polar residues and reduces this difference to 29 nm. The present study expands our knowledge concerning the role of the reorganization of the internal H-bond network for color tuning in general and provides a detailed investigation of the tuning mechanism in phR in particular.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo.

The Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in trans that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the trans-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus. IMPORTANCE We revealed the mechanism that explains how the NgAgo eliminates the invading foreign DNA and bacteriophage in bacterial cells at 37°C, and by leveraging this discovery, NgAgo can be programmed to target a plasmid or a chromosome locus.

The signal transfer from the receptor NpSRII to the transducer NpHtrII is not hampered by the D75N mutation.

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.

Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis.

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ∼500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.

An amino acid residue (S201) in the retinal binding pocket regulates the photoreaction pathway of phoborhodopsin.

Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (λ(max)) at 482 nm and can thermally revert back to spR above -160 °C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9═C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.

Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets.

Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5' phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3' of the target RNA which is recognized by 5' sequence of the gDNA. These results confirmed that NgAgo-gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3' of the target RNA, which is a new finding that has not been reported in the past.

Protein-protein interaction changes in an archaeal light-signal transduction.

Negative phototaxis in Natronomonas pharaonis is initiated by transient interaction changes between photoreceptor and transducer. pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) and the cognate transducer protein, pHtrII, form a tight 2 : 2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. We have studied the signal transduction mechanism in the ppR/pHtrII system by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. In the paper, spectral comparison in the absence and presence of pHtrII provided fruitful information in atomic details, where vibrational bands were identified by the use of isotope-labeling and site-directed mutagenesis. From these studies, we established the two pathways of light-signal conversion from the receptor to the transducer; (i) from Lys205 (retinal) of ppR to Asn74 of pHtrII through Thr204 and Tyr199, and (ii) from Lys205 of ppR to the cytoplasmic loop region of pHtrII that links Gly83.

Reaction dynamics of halorhodopsin studied by time-resolved diffusion.

Reaction dynamics of a chloride ion pump protein, halorhodopsin (HR), from Natronomonas pharaonis (N. pharaonis) (NpHR) was studied by the pulsed-laser-induced transient grating (TG) method. A detailed investigation of the TG signal revealed that there is a spectrally silent diffusion process besides the absorption-observable reaction dynamics. We interpreted these dynamics in terms of release, diffusion, and uptake of the Cl(-) ion. From a quantitative global analysis of the signals at various grating wavenumbers, it was concluded that the release of the Cl(-) ion is associated with the L2 --> (L2 (or N) <==> O) process, and uptake of Cl(-) occurs with the (L2 (or N) <==> O) -->NpHR' process. The diffusion coefficient of NpHR solubilized in a detergent did not change during the cyclic reaction. This result contrasts the behavior of many photosensor proteins and implies that the change in the H-bond network from intra- to intermolecular is not significant for the activity of this protein pump.

Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction.

We report an atomic-resolution structure for a sensory member of the microbial rhodopsin family, the phototaxis receptor sensory rhodopsin II (NpSRII), which mediates blue-light avoidance by the haloarchaeon Natronobacterium pharaonis. The 2.4 angstrom structure reveals features responsible for the 70- to 80-nanometer blue shift of its absorption maximum relative to those of haloarchaeal transport rhodopsins, as well as structural differences due to its sensory, as opposed to transport, function. Multiple factors appear to account for the spectral tuning difference with respect to bacteriorhodopsin: (i) repositioning of the guanidinium group of arginine 72, a residue that interacts with the counterion to the retinylidene protonated Schiff base; (ii) rearrangement of the protein near the retinal ring; and (iii) changes in tilt and slant of the retinal polyene chain. Inspection of the surface topography reveals an exposed polar residue, tyrosine 199, not present in bacteriorhodopsin, in the middle of the membrane bilayer. We propose that this residue interacts with the adjacent helices of the cognate NpSRII transducer NpHtrII.