The oligodeoxynucleotide sequences corresponding to never-expressed peptide motifs are mainly located in the non-coding strand

<p>Abstract</p> <p>Background</p> <p>We study the usage of specific peptide platforms in protein composition. Using the pentapeptide as a unit of length, we find that in the universal proteome many pentapeptides are heavily repeated (even thousands of times), whereas some are quite rare, and a small number do not appear at all. To understand the physico-chemical-biological basis underlying peptide usage at the proteomic level, in this study we analyse the energetic costs for the synthesis of rare and never-expressed versus frequent pentapeptides. In addition, we explore residue bulkiness, hydrophobicity, and codon number as factors able to modulate specific peptide frequencies. Then, the possible influence of amino acid composition is investigated in zero- and high-frequency pentapeptide sets by analysing the frequencies of the corresponding inverse-sequence pentapeptides. As a final step, we analyse the pentadecamer oligodeoxynucleotide sequences corresponding to the never-expressed pentapeptides.</p> <p>Results</p> <p>We find that only DNA context-dependent constraints (such as oligodeoxynucleotide sequence location in the minus strand, introns, pseudogenes, frameshifts, etc.) provide a coherent mechanistic platform to explain the occurrence of never-expressed versus frequent pentapeptides in the protein world.</p> <p>Conclusions</p> <p>This study is of importance in cell biology. Indeed, the rarity (or lack of expression) of specific 5-mer peptide modules implies the rarity (or lack of expression) of the corresponding <it>n</it>-mer peptide sequences (with <it>n </it>< 5), so possibly modulating protein compositional trends. Moreover the data might further our understanding of the role exerted by rare pentapeptide modules as critical biological effectors in protein-protein interactions.</p

TTF-1 Action on the Transcriptional Regulation of Cyclooxygenase-2 Gene in the Rat Brain

We have recently found that thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, is postnatally expressed in discrete areas of the hypothalamus and closely involved in neuroendocrine functions. We now report that transcription of cyclooxygenase-2 (COX-2), the rate limiting enzyme in prostaglandin biosynthesis, was inhibited by TTF-1. Double immunohistochemistry demonstrated that TTF-1 was expressed in the astrocytes and endothelial cells of blood vessel in the hypothalamus. Promoter assays and electrophoretic mobility shift assays showed that TTF-1 inhibited COX-2 transcription by binding to specific binding domains in the COX-2 promoter. Furthermore, blocking TTF-1 synthesis by intracerebroventricular injection of an antisense oligomer induced an increase of COX-2 synthesis in non-neuronal cells of the rat hypothalamus, and resulted in animals' hyperthermia. These results suggest that TTF-1 is physiologically involved in the control of thermogenesis by regulating COX-2 transcription in the brain

Delivery of small nucleic acids by conjugation to carbohydrates and lipids as novel research and therapeutic tools

399 p. : il., graf.Small nucleic acids present a great potential to silence specific genes or inhibit the biological activity of specific proteins. Unfortunately, their poor cellular accessibility presents a big challenge for developing an efficient delivery system. In this regard, conjugation of appropriate molecules to small nucleic improves their pharmacokinetic behaviours and cellular uptake efficiencies, endowing them with entirely new properties. In the present work different carbohydrate- and lipid-oligonucleotide conjugates have been studied as novel tools for targeted delivery and enhancement of cellular permeability. The results obtained in this work indicate that keeping a certain distance between DNA and sugar modification could be important for a better incorporation of this type of conjugates into the target cell. Also long or double-tailed lipid modifications are preferred for an enhanced incorporation of lipid-oligonucleotide conjugates into membrane-model and cell systems.The present thesis has been performed Departamento de Bioquímica y Biología Molecular de la Universidad del País Vasco and Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU). This work has been supported mainly by CSIC (CARBINH, PIF06-045). The author received a research fellowship from the University of the Basque Country (PIFA01/2006/052, June 2007-May 2011) and a research contract supported by Fundación Biofísica Bizkaia (June 2011-May 2012)

Antisense oligonucleotide-mediated therapeutic strategies for neurodegenerative repeat expansion diseases

Over 40 diseases, primarily affecting the nervous system, are caused by expansion of simple repetitive sequences found throughout the human genome, termed repeat expansion diseases. Expansions can occur in coding and non-coding regions of the genome, leading to several proposed mechanisms of disease, accumulation of either toxic RNA or toxic protein, although gain-of-function mechanisms are suggested causes of pathogenesis. Currently, there is no cure nor effective treatment strategy for any repeat expansion diseases. However, for many of these expansion diseases, splice-switching antisense oligonucleotides (AOs) may offer promise as a therapeutic strategy, as these compounds have already demonstrated efficacy in the treatment of other types of genetic disorders. Antisense oligonucleotides are short synthetic nucleic acid analogues, designed to target specific pre-mRNA sequences by reverse-complementary Watson-Crick binding, thereby modifying processing and/or abundance of the transcript and the sequence of the encoded protein. While there are a number of applications for AOs, this study focuses on their utility for preventing translation of toxic protein isoforms, either by altering the target transcript to encode a truncated protein isoform, or by disrupting the reading frame to downregulate endogenous protein production. The first part of this study focused on ameliorating the toxic polyglutamine tract found in the ataxin-3 protein that causes spinocerebellar ataxia type 3 (SCA3). One of nine known polyglutamine disorders, SCA3 is a clinically heterogeneous disease, primarily exemplified by progressive ataxia impairing the speech, balance and gait of affected individuals. SCA3 is caused by expansion of a glutamine-encoding tract located at the 5′ end of the penultimate exon (exon 10) of the ATXN3 gene transcript, resulting in conformational changes in ataxin-3 and a toxic gain-of-function. Here, we describe highly efficient removal of the toxic polyglutamine tract of ataxin-3 in vitro by phosphorodiamidate morpholino oligomers (PMOs). Additionally, these PMOs induced a potentially beneficial downregulation of both the expanded iv and non-expanded protein isoforms. As SCA3 has a typical age of onset in the fourth decade, the observed downregulation could delay age of onset by reducing the amounts of the toxic aggregates. Although we induce downregulation of both isoforms, we believe that the proportion of the truncated protein may be sufficient for overall function of ataxin-3, as some studies have shown ataxin-3 protein to be partially dispensable. Recently, several in vitro and in vivo studies have found that targeted knockdown of transcription elongation factors SUPT4H1, and to a lesser extent SUPT5H, can reduce aggregation of expanded transcripts and protein, and alleviate the disease phenotype in animal models of various expansion diseases. We therefore sought to investigate in vitro, the potential of AO-mediated SUPT4H1 downregulation as a therapeutic strategy. We found that our AOs were able to significantly downregulate SUPT4H1, with minimal changes to the rest of the transcriptome. We then assessed whether this downregulation of SUPT4H1 lead to a reduction in expanded ATXN3 mRNA and/or ATXN3 protein expression, however, unfortunately in the models available and under the current study, no modification to the ATXN3 transcript or protein was observed. This lack of effect may be due to the relatively short, expanded repeat lengths in SCA3 cell lines, and we therefore recommend that future studies assess genes with larger expansions, such as the 100-1000s repeat tracts frequently observed in myotonic dystrophy type 1 (DMPK). In order to create an efficient screening process for finding clinic-ready AOs, it is important to have a detailed understanding of the principles of AO design. We therefore present a comprehensive rationale for efficiently design and in vitro delivery of splice modulating AOs. These approaches and recommendations provide a streamlined methodology for any researcher developing AO therapeutics. The results presented in this thesis indicate that morpholino oligomers will provide superior benefit for the treatment of spinocerebellar ataxia type 3, without the toxic effects that result from other antisense oligomer chemistries. Additionally, AO-induced SUPT4H1 knockdown may yet demonstrate therapeutic v application for a multitude of expansion diseases, pending further investigation into the whole transcriptome effects and in vivo efficacy of this strategy. Lastly, our guidelines for therapeutic AO development should aid other researchers in creating the most efficacious and safe AOs for clinical trials. The work presented in this thesis contributes to the greater body of knowledge about the applications of AOs, as well as the need for reliable and systematic protocols in AO research and interpretation. With ongoing collaboration from our industry partners, Sarepta Therapeutics, there is hope that the work presented here will provide a solid foundation for further research into AO therapeutics for the treatment of neurodegenerative expansion diseases

Thermostable DNA polymerases in replication, repair and biotechnology

PhD ThesisMany archaea contain a unique DNA polymerase, DNA Pol D. This enzyme is a heterodimer composed of a large subunit (polymerase) and a small subunit (3’- 5’ proof reading exonuclease). The enzyme from Pyroccocus furiosus is inhibited by the presence of uracil in template strands. This research has shown that a single uracil located as far as 134 bases ahead of the primertemplate junction causes inhibition of replication. Further, using replication fork mimics, it is shown that, as expected, uracil on a template strand being copied by Pol D causes inhibition. Surprisingly, though, the presence of uracil on a complementary non-copied strand is also inhibitory. A model for uracil recognition by Pol D is proposed. The biochemical properties of the individual, large and small, subunits of the Pol D heterodimer were analysed. Both subunits were found to possess activity when expressed alone although the activity was greatly reduced compared to the Pol D heterodimer. It was not possible to regain the level of activity observed in the Pol D holoenzyme by mixing the two subunits in vitro. This finding contributed to the hypothesis that the carboxyl-terminal region of the large subunit contains an Fe-S cluster that is lost when the protein is purified aerobically. Attempts were made to express Pol D in archaeal hosts and purify the protein with the correct metallo-status; regrettably, these were not successful. Two thermostable bacterial family-B (pol II) DNA polymerases were cloned and expressed in E.coli and their biochemical properties analysed. The enzymes were found to possess many properties that make them amenable to biotechnology: polymerase activity, 3’-5’ proofreading activity, high fidelity rates and the ability to bypass uracil located in template strand DNA. Unfortunately, thermostability assays revealed that the polymerases denatured on exposure to temperatures ~85°C, making them unsuitable in the PCR. Thus, further manipulation is required to determine whether the polymerases have applications in biotechnology

Oligonucleotide therapies for RNA and DNA : modulation of natural splice-variants, DNA structure & characterization of new synthetic nucleotides and reporter cell lines

Oligonucleotide therapy is an evolving field having shown fast and important developments in the last years. From genetics to metabolic, inflammatory, immunodeficiency diseases, cancer and viral infections the medical applications for this type of therapy are becoming broader every day. However, the major challenge for these therapies is still the delivery; thus new chemistries, as well as improved delivery vectors are needed. Equally, the lack of relevant reporter systems hampers the characterization and progress of these emergent “drugs.” This thesis work was aimed to address some of the gaps presented above. Thus, the introduction section starts with a brief overview of the gene therapy. It continues with an explanation of the oligonucleotide therapy strategies and technologies used for RNA and DNA targeting and ends with a clinical case for each approach. The methodology section explains the theoretical and practical aspects of the most relevant techniques in this study and the results section explain the rational and gives a brief presentation of the main results and conclusions for each paper. Paper I presents a new splice-switching approach for treating diseases by regulating proteins at the splicing level. The new principle was to convert the normal splice form to a natural, but less abundant and inactive, splice variant to treat hypercholesterolemia. Paper II shows the development and characterization of a new collection of reporter cell lines for splice- switching. These reporter cell lines can serve as models for cell-type-specific screenings of different ON chemistries and delivery vectors. Paper III and IV demonstrate the development and characterization of a new nucleic acid chemical modification providing an oligomer with cell penetrating properties. Finally, in paper V a new mechanism for gene expression regulation at the genome level is presented

Nullomer Derived Anticancer Peptides (NulloPs): Differential Lethal Effects on Normal and Cancer Cells \u3cem\u3ein vitro\u3c/em\u3e

We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in1M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time

Development and Evaluation of Nano‑scale Systems for Targeted Delivery to Treat Liver Fibrosis

Excessive deposition of ECM is the common characteristic of liver fibrosis. During hepatic fibrosis, various inflammatory cytokines are released and trigger the activation of quiescent HSCs. The activated HSCs play the major role in producing extra amount collagen. It becomes very crucial to focus on HSCs to find out therapeutics, such as inhibiting collagen synthesis, inhibiting activation to myofibroblasts, or controlling inflammation. To control excessive collagen synthesis, one triplex forming oligonucleotides (TFO), was systemically administrated to prevent type I collagen mRNA transcription. To enhance circulation time and targeted delivery efficiency, TFO was conjugated to M6P‑HPMA and showed efficient targeted delivery to HSCs. Two week short term in vivo i.v. administration also showed the therapeutic effects on liver fibrosis by M6P‑HPMA‑TFO. Transforming growth factor β1 (TGF‑β1) acts as the initial factor for liver fibrosis. TGF‑β1 gene was demonstrated to be interfered by siRNA in a sequence and dose dependent mode in HSC‑T6 cell line. Later, GFAP promoter driven HSC‑specific pri‑miRNA mimic and pri‑miRNA cluster mimic showed HSC‑specific TGF‑β1 gene silencing to avoid nonspecific inhibition of TGF‑β1 expression in other cells and organs. The novel LPA antagonist, PTP, can interact with LPA receptor on the surface of cells to inhibit the proliferation, which is also one consequence of HSCs activation. However, the low aqueous solubility affects its in vivo application. Therefore, poly(ethylene glycol)‑b‑poly(carbonate‑co‑lactide) copolymers were used to make micellar formulation to enhance solubility. PEG‑PCcL micelles were applied to increase the aqueous solubility of PTP. In vivo administration of PTP loaded PEG‑PCcL showed therapeutic effects on fibrosis in common bile duct ligated mice

Isolation And Analysis Of Peptides Binding To Helix 69 Of E. Coli 23 S Rrna From M13 Phage Display

Peptides binding to helix 69 of domain IV or residues 1906-1924, of 23 S rRNA of E.coli were being selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through four rounds was being followed. After sequencing phage clones of the fourth round, two peptide sequences dominated the phage pool, STYTSVS and NQVANHQ. The later sequence was a unique sequence since this sequence contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. Phage-display methodology demonstrated the rapid feasibility of identification, and isolation of small peptides that bind to 23 S rRNA in an effort to discover new RNA-binding motifs that have potential therapeutic applications. For evaluating the preliminary binding affinity of these peptides with H69, fluorescence assays were applied. For this assay, the fluorescence intensity of the NQVANHQ Tentagel beads was observed to be higher than STYTSVS Tentagel beads indicating that peptide NQVANHQ is having higher affinity for H69 as compared to STYTSVS peptide. But the higher binding affinity of the NQVANHQ peptide was further validated with more sensitive method of electrospray ionization (ESI) mass spectroscopy. The apparent dissociation constant (Kd) obtained for H69 and NQVANHQ-NH2 peptide was in the low micromolar range (11 µM). This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA (1 to 10 µM). The ESI-MS experiments with H69 variant UUU RNA and peptide NQVANHQ-NH2 gave the relative dissociation constant (Kd) at 1:1 stoichiometry as 19 µM. The higher value of Kd for this complex revealed that the presence of all three pseudouridine residues positively contributed towards binding of this peptide to H69. Consecutively, to learn about the role of individual pseudouridines at position 1911 and 1915 towards binding of the peptide, the ESI-MS experiments were performed with two H69 variants,UYY and YUY. The apparent dissociation constants (Kd) for the 1:1 complex for these two RNAs decreased by 2.5-fold showing that peptide binding site is located at or near the loop region containing the pseudouridines at positions 1911 and 1915. In addition, the effect of pH on the complex formation of H69 and UUU RNA with NQVANHQ-NH2 peptide was studied at two different pH values of 7.0 and 5.2. There was three-fold decrease of the apparent dissociation constant for the 1:1 complex of RNA and the peptide indicating that either protonation of the RNA or the peptide structure influenced this change in binding of the two species. The specificity of the peptide for H69 was tested with related RNA such as human H69 and unrelated RNAs such as helix 31 and A-site rRNA. The peptide showed three-fold lower affinity than the target H69 RNA for all these RNAs suggesting that the peptide has features for developing it as a lead compound for novel antimicrobial