Immobilization of different biomolecules by atomic force microscopy

<p>Abstract</p> <p>Background</p> <p>Micrometer resolution placement and immobilization of probe molecules is an important step in the preparation of biochips and a wide range of lab-on-chip systems. Most known methods for such a deposition of several different substances are costly and only suitable for a limited number of probes. In this article we present a flexible procedure for simultaneous spatially controlled immobilization of functional biomolecules by molecular ink lithography.</p> <p>Results</p> <p>For the bottom-up fabrication of surface bound nanostructures a universal method is presented that allows the immobilization of different types of biomolecules with micrometer resolution. A supporting surface is biotinylated and streptavidin molecules are deposited with an AFM (atomic force microscope) tip at distinct positions. Subsequent incubation with a biotinylated molecule species leads to binding only at these positions. After washing streptavidin is deposited a second time with the same AFM tip and then a second biotinylated molecule species is coupled by incubation. This procedure can be repeated several times. Here we show how to immobilize different types of biomolecules in an arbitrary arrangement whereas most common methods can deposit only one type of molecules. The presented method works on transparent as well as on opaque substrates. The spatial resolution is better than 400 nm and is limited only by the AFM's positional accuracy after repeated z-cycles since all steps are performed in situ without moving the supporting surface. The principle is demonstrated by hybridization to different immobilized DNA oligomers and was validated by fluorescence microscopy.</p> <p>Conclusions</p> <p>The immobilization of different types of biomolecules in high-density microarrays is a challenging task for biotechnology. The method presented here not only allows for the deposition of DNA at submicrometer resolution but also for proteins and other molecules of biological relevance that can be coupled to biotin.</p

DNA-nanostructure-assembly by sequential spotting

<p>Abstract</p> <p>Background</p> <p>The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabrication. Thus we combined the capabilities of molecular ink lithography with DNA-self-assembling capabilities to arrange DNA in an independent array which allows addressing molecules in nanoscale dimensions.</p> <p>Results</p> <p>For the construction of DNA based nanostructures a method is presented that allows an arrangement of DNA strands in such a way that they can form a grid that only depends on the spotted pattern of the anchor molecules. An atomic force microscope (AFM) has been used for molecular ink lithography to generate small spots. The sequential spotting process allows the immobilization of several different functional biomolecules with a single AFM-tip. This grid which delivers specific addresses for the prepared DNA-strand serves as a two-dimensional anchor to arrange the sequence according to the pattern. Once the DNA-nanoarray has been formed, it can be functionalized by PNA (peptide nucleic acid) to incorporate advanced structures.</p> <p>Conclusions</p> <p>The production of DNA-nanoarrays is a promising task for nanobiotechnology. The described method allows convenient and low cost preparation of nanoarrays. PNA can be used for complex functionalization purposes as well as a structural element.</p

The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing

The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes

Mammalian WDR12 is a novel member of the Pes1–Bop1 complex and is required for ribosome biogenesis and cell proliferation

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1–Bop1–WDR12 in yeast (Nop7p–Erb1p–Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells

C‐reactive protein flare‐response predicts long‐term efficacy to first‐line anti‐PD‐1‐based combination therapy in metastatic renal cell carcinoma

Objectives Immune checkpoint blockade (IO) has revolutionised the treatment of metastatic renal cell carcinoma (mRCC). Early C-reactive protein (CRP) kinetics, especially the recently introduced CRP flare-response phenomenon, has shown promising results to predict IO efficacy in mRCC, but has only been studied in second line or later. Here, we aimed to validate the predictive value of early CRP kinetics for 1st-line treatment of mRCC with αPD-1 plus either αCTLA-4 (IO+IO) or tyrosine kinase inhibitor (IO+TKI). Methods In this multicentre retrospective study, we investigated the predictive potential of early CRP kinetics during 1st-line IO therapy. Ninety-five patients with mRCC from six tertiary referral centres with either IO+IO (N = 59) or IO+TKI (N = 36) were included. Patients were classified as CRP flare-responders, CRP responders or non-CRP responders as previously described, and their oncological outcome was compared. Results Our data validate the predictive potential of early CRP kinetics in 1st-line immunotherapy in mRCC. CRP responders, especially CRP flare-responders, had significantly prolonged progression-free survival (PFS) compared with non-CRP responders (median PFS: CRP flare-responder: 19.2 months vs. responders: 16.2 vs. non-CRP responders: 5.6, P < 0.001). In both the IO+IO and IO+TKI subgroups, early CRP kinetics remained significantly associated with improved PFS. CRP flare-response was also associated with long-term response ≥ 12 months. Conclusions Early CRP kinetics appears to be a low-cost and easy-to-implement on-treatment biomarker to predict response to 1st-line IO combination therapy. It has potential to optimise therapy monitoring and might represent a new standard of care biomarker for immunotherapy in mRCC

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1

Rapid conditional knock-down–knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down–knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein