Nucleic Acid Binding Domain Research Articles

Crystal structure of TbAlba1 from Trypanosoma brucei

Alba (Acetylation lowers binding affinity) domain is a small, dimeric nucleic acid-binding domain, which is widely distributed in archaea and numbers of eukaryotes. Alba domain containing proteins have been reported to be involved in many cellular processes, such as regulation of translation, maintaining genome stability, regulation of RNA processing and so on. In Trypanosoma brucei (T. brucei), there are four Alba proteins identified, which are named TbAlba1 to TbAlba4. However, the structure and function of TbAlba proteins are still unknown. Here, we solved the crystal structure of TbAlba1 to a resolution of 2.46 Å. TbAlba1 adopts a similar Alba-fold, which comprises of four β-strands (β1-β4) and three long α-helices (α1-α3). Furthermore, TbAlba1 displays some structural features quite different from other Alba proteins. These differences may imply the diverse biological roles of Alba family members.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase

The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation–seq, and cross-linking immunoprecipitation–seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

Cell division requires RNA eviction from condensing chromosomes.

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

1H, 13C, and 15N backbone chemical shift assignments of the nucleic acid-binding domain of SARS-CoV-2 non-structural protein 3e

The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus’ proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex

SARS-CoV-2 is the causative agent of the 2019–2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer

Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.

Neofunctionalized gene RTl3 and SOX9 collectively modulate COL2A1 expression in human articular chondrocytes

Purpose: Retrotransposon-derived DNA is a known source of genetic variation, but the biological significance of these molecules in chondrocytes, the resident cell type present in cartilage, is still unknown. A mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 was found to be differentially expressed in human OA cartilage vs normal samples but the function of RTL3 in cartilage remained unexplored. The goal of our study was to determine the functional role of the mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 in human chondrocytes. Methods: These studies were approved by the institutional review boards (IRBs) of Northeast Ohio Medical University and Summa Health Systems (Akron, OH) prior to the initiation of the studies. Chondrocytes were isolated by sequential enzymatic digestion of discarded and de-identified cartilage samples from patients undergoing total joint arthroplasty. Total RNA was isolated using Trizol. mRNA expression was assessed via Taqman assays and protein expression by immunoblotting using validated antibodies. Human RTL3 and SOX-9 expression was depleted using validated siRNAs (GE Dharmacon ONTARGETplus) and X-tremeGENE siRNA Transfection Reagent (SigmaAldrich). For ectopic expression, human chondrocytes were transfected with a RTL3 expression plasmid (Origene) using X-tremeGENE HP DNA Transfection Reagent (SigmaAldrich). COL2A1 promoter reporter assays employed a pCOL2A1-luciferase reporter (GeneCopoeia) and the luciferase activity normalized with a Renilla reporter (Promega). Statistical analyses were conducted using student t-tests or one-way analysis of variation (ANOVA) with Tukey’s post-hoc tests. Results: RTL3 contains a nucleic acid binding domain so we first determined if RTL3 was directly involved in regulating the expression of chondrocyte signature genes expression through gain of function and loss of function analyses. Upon RTL3 depletion, COL2A1 mRNA and protein levels were significantly reduced and activity of the COL2A1 promoter reporter was also significantly decreased. Transfection with an RTL3 expression plasmid under the control of the CMV promoter significantly increased the COL2A1 mRNA and protein levels and enhanced COL2A1 promoter reporter activity was observed in a dose-dependent manner. Together,these results indicated that RTL3 has a direct role in regulating chondrocyte expression of COL2A1. To determine a possible mode of action, we determined if RTL3 modulates COL2A1 expression via known COL2A1 regulator SOX-9. SOX-9 depletion reduced expression of COL2A1 as well as COL2A1 promoter reporter activity. When RTL3 was depleted, constitutive expression of SOX-9 mRNA and protein were significantly reduced compared to controls and the activity of the COL2A1 promoter reporter was also inhibited. Conversely, over-expression of RTL3 was able to significantly enhance mRNA and protein levels of SOX-9 in chondrocytes and increase COL2A1 promoter reporter activity in vitro. Interestingly, in chondrocytes with SOX-9 knockdown and RTL3 overexpression, COL2A1 promoter activity was half that of cells expressing endogenous SOX-9. These results indicate RTL3 or SOX9 alone are not sufficient for optimal expression of COL2A1 but both act in concert to to regulate COL2A1 expression in chondrocytes. Conclusions: The mammalian retrotransposon RTL3 has lost the ability to retrotranspose but has retained an open reading frame suggestive of new cellular functions. Here, we utilized loss-of-function and gain-of-function approaches to demonstrate a previously unknown role of the mammalian retrotransposon transcript RTL3 in the regulation of COL2A1 in human chondrocytes in vitro. siRNA-depletion of RTL3 reduced constitutive SOX-9 expression, pCOL2A1 promoter reporter activity and COL2A1 transcription. Forced expression of RTL3 rescued these effects. SOX-9 siRNA-depletion and RTL3 over expression experiments elucidated that expression of both RTL3 and SOX-9 are important for COL2A1 regulation in chondrocytes. The details of this previously unknown mechanism of regulation warrants further study.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

NPM1 exhibits structural and dynamic heterogeneity upon phase separation with the p14ARF tumor suppressor.

Nucleophosmin (NPM1) is an abundant nucleolar protein that aids in the maturation of pre-ribosomal particles and participates in oncogenic stress responses through its interaction with the Alternative Reading Frame tumor suppressor (p14ARF). NPM1 mediates multiple mechanisms of phase separation which contribute to the liquid-like properties of nucleoli. However, the effects of phase separation on the structure and dynamics of NPM1 are poorly understood. Here we show that NPM1 undergoes phase separation with p14ARF in vitro, forming condensates that immobilize both proteins. We probed the structure and dynamics of NPM1 within the condensed phase using solid-state NMR spectroscopy. Our results demonstrate that within the condensed phase, the NPM1 oligomerization domain forms an immobile scaffold, while the central intrinsically disordered region and the C-terminal nucleic acid binding domain exhibit relative mobility.

Elucidating the functional aspects of different domains of bean common mosaic virus coat protein

The coat protein (CP) is the only structural protein present in the polyprotein of bean common mosaic virus. The well known characteristics of the CP are self-oligomerization and nucleic acid binding activity. The studies of the coat protein mutants revealed that the oligomeric property of CP solely depends on the amino-terminal residues and the nucleic acid binding domain present at the 194–202 residue position. The 3′UTR RNA of the virus showed high binding affinity with the RNA binding domain as compared to the 5′UTR RNA. Further, the intrinsic fluorescence study of the CP also suggested that the N- and C-terminal of CP contains a highly disordered region. The present study also illustrates that the coat protein contains a conserved RNA binding pocket among the potyviruses, but displays divergent oligomerization propensities due to the difference in residue at the N- and C-terminal.

Abstract 4114: NPM1 upregulates the transcription of PD-L1 and suppresses T-cell activity in triple negative breast cancer

Abstract Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here we verified that triple-negative breast cancer (TNBC) had higher PD-L1 expression than other subtypes. We then discovered that nucleophosmin (NPM1) bound to PD-L1 promoter specifically in TNBC cells and activated PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrated that PARP1 suppressed PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which was required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevated PD-L1 expression in TNBC and exerted a synergistic effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a novel transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy. Citation Format: Qian Long, Yizhuo Li, Ge Qin, Changlin Zhang, Dingbo Shi, Miao Chen, Shuihan Shi, Kefang Zhang, Wuguo Deng. NPM1 upregulates the transcription of PD-L1 and suppresses T-cell activity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4114.

The splicing factor U2AF1 contributes to cancer progression through a noncanonical role in translation regulation.

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias, and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid-binding domain, recognition of the 3' splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we uncovered a noncanonical function of U2AF1, showing that it directly binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysome profiling indicates that the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8, which contributes to metastasis, inflammation, and cancer progression in mice and humans.

Activated YAP causes renal damage of type 2 diabetic nephropathy.

Yes-associated protein (YAP) is a critical factor of Hippo pathway. It can control organ size, regulate proliferation, differentiation, and apoptosis of cells, and mediate epithelial-mesenchymal transition and cell contact inhibition. It has gradually become a hot spot in the field of diabetic nephropathy (DN). Tea domain (TEAD) is a factor with a deoxyribose nucleic acid (DNA) binding domain, which combines with activated YAP to control the expression of their important target factor - connective tissue growth factor (CTGF). We have investigated the role of YAP in type 2 diabetic nephropathy and evaluated the correlation between YAP and the progress of type 2 diabetic nephropathy. We have detected the expression of YAP, TEAD and CTGF in normal people (n=10) and patients with DN (n=51) by immunohistochemical and immunofluorescence staining and evaluated the relationship among clinical, pathologic data and YAP expression in type 2 diabetic nephropathy. In kidneys of type 2 diabetic nephropathy, YAP, TEAD and CTGF were highly expressed in the nucleus of glomerular podocytes. In those healthy kidneys, however, all three of the above factors were mainly expressed in cytoplasm. Furthermore, the high expression of YAP in DN had relevance to increasing systolic blood pressure (SBP) (r=0.484, p=0.019), blood urea nitrogen (BUN) (r=0.522, p=0.032), creatinine (Cr) (r=0.496, p=0.031), progression of DN stage (r=0.647, p=0.001) and progression of DN pathologic classification (r=0.298, p=0.033). In addition, decreasing serum albumin (SAlb) (r=-0.656, p=0.001) and estimated glomerular filtration rate (eGFR) (r=-0.607, p=0.006) were also correlated with the high expression of YAP in DN. High expression of YAP, TEAD and CTGF in kidney tissues suggested that YAP played a significant role in the renal damage of type 2 diabetic nephropathy. YAP that is correlated with SBP, BUN, Cr, DN stage, DN pathologic classification, SAlb and eGFR, suggested that inhibition of the activity of YAP might have the effect in delaying DN progression.

Contribution of the cold shock protein CspA to virulence in Xanthomonas oryzae pv. oryzae.

Summary Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid‐binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA–CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA‐binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single‐deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real‐time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

Systematic miRNome profiling reveals differential microRNAs in transgenic maize metabolism

BackgroundWhile some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks.ResultsThirteen different conserved miRNAs were found to be dys-regulated in GM samples. The insecticide Bt GM variety had the most distinct miRNome. These miRNAs target a range of endogenous transcripts, such as transcription factors and nucleic acid binding domains, which play key molecular functions in basic genetic regulation. In addition, we have identified 20 potential novel miRNAs with target transcripts involved in lipid metabolism in maize. isomiRs were also found in 96 conserved miRNAs sequences, as well as potential transgenic miRNA sequences, which both can be a source of potential off-target effects in the plant genome. We have also provided information on technical limitations and when to carry on additional in vivo experimental testing.ConclusionsThese findings do not reveal hazards per se but show that robust and reproducible miRNA profiling technique can strengthen the assessment of risk by detecting any new intended and unintended dsRNA molecules, regardless of the outcome, at any stage of GMO development.

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA–SMN interactions. Our results reveal that motifs that drive RNA–SMN interactions are diverse and suggest that tight RNA–SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA–SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.

Z-DNA binding protein 1 (ZBP1) is an innate sensor of Influenza vRNPs and activates programmed cell death and assembly of NLRP3 inflammasome

Abstract Influenza A virus (IAV) infection triggers pulmonary epithelial cell death and inflammation which promote bronchioalveolar tissue damage and failure of lung function. However, the mechanism of IAV induced cell death and inflammation remains unclear. RIP homotypic interaction motif (RHIM) has evolved to play critical role in cell death and inflammation. Z-DNA binding protein 1 (ZBP1/DAI) is unique among RHIM-containing proteins since it has additional Za nucleic acid binding domains. Although ZBP1 domain architecture suggests its function as nucleic acid sensor, its precise physiological role is not established. We discovered that ZBP1 is a central activator of IAV induced programmed cell death and NLRP3 inflammasome activation in both immune and non-immune cells. Mechanistically, ZBP1 senses ribonucleoprotein complexes (vRNPs) of IAV and engage RIPK3-caspase-8 dependent apoptosis, necroptosis and NLRP3 inflammasome assembly. ZBP1 also drives RIPK1 dependent secretion of TNF and IL-6 cytokines after IAV infection. We also discovered that apical activation of RIG-I/MAVS/IFNAR signaling licenses ZBP1 upregulation and activation. In contrast to other RNA viruses, IAV replicates inside the nucleus. Accordingly, we observed localization of ZBP1 in the nucleus suggesting its role in sensing IAV inside the nucleus. Strikingly, we also found that ZBP1 undergoes ubiquitination in response to IAV infection to assemble cell death signaling complexes. In IAV infected mice, ZBP1 induces cell death in lung epithelial cells and its absence conferred better survival advantage, despite high viral titers in the lung. Overall, our work demonstrates the central role of ZBP1 in sensing IAV infection to trigger cell death and inflammation.

Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides.

Antisense oligonucleotides (ASOs) are versatile tools that can regulate multiple steps of RNA biogenesis in cells and living organisms. Significant improvements in delivery, potency, and stability have been achieved through modifications within the oligonucleotide backbone, sugar and heterocycles. However, these modifications can profoundly affect interactions between ASOs and intracellular proteins in ways that are only beginning to be understood. Here, we report that ASOs with specific backbone and sugar modifications can become localized to cytoplasmic ribonucleoprotein granules such as stress granules and those seeded by the aggregation of specific ASO-binding proteins such as FUS/TLS (FUS) and PSF/SFPQ (PSF). Further investigation into the basis for ASO-FUS binding illustrated the importance of ASO backbone and hydrophobic 2′ sugar modifications and revealed that the C-terminal region of FUS is sufficient to retain ASOs in cellular foci. Taken together, the results of this study demonstrate that affinities of various nucleic acid binding domains for ASO depend on chemical modifications and further demonstrate how ASO–protein interactions influence the localization of ASOs.

Polyvalent Proteins, a Pervasive Theme in the Intergenomic Biological Conflicts of Bacteriophages and Conjugative Elements.

ABSTRACTIntense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular “weaponry” deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance.IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems.

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics. Although, the siRNA constructs have already given very promising results in the cancer therapy, the in vivo application of RNA-based oligonucleotide therapeutics still is limited due to their sensitivity to serum nucleases and some toxicity. We propose a carrier for RNA-based therapeutics that is made of bioengineered spider silk. We showed that functionalized bioengineered spider silk spheres not only protected RNA-based therapeutics from degradation by serum nucleases, but what is more important the embedding of siRNA into silk spheres delayed and extended target gene silencing compared with naked oligonucleotides. Moreover, we showed that plain silk spheres did not have unspecific effect on target gene levels proving not only to be non-cytotoxic but also very neutral vehicles in terms of TLR9/STAT3 activation in macrophages. We demonstrated advantages of novel delivery technology in safety and efficacy comparing with delivery of naked CpG-STAT3siRNA therapeutics.