Browsing by Subject "Oligonucleotides"

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Nuclear Behaviors of ERK1/2 Signaling
(2017-04-18) Karra, Aroon Saikumar; D'Orso, Iván; Cobb, Melanie H.; Conrad, Nicholas; Pearson, Gray W.
The rat sarcoma (Ras)- rapidly accelerated fibrosarcoma (Raf)- mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) kinase (MEK)- ERK pathway is essential for proper development and homeostatic regulation in eukaryotic cells. Many pathway functions are carried out by ERK1 and ERK2 (ERK1/2), serine/threonine protein kinases that interact with a large number of substrates in several cell compartments. ERK1/2 are crucial for major cellular phenomena such as proliferation, differentiation, and programmed cell death. Despite a large body of knowledge about this pathway, a clear understanding of how specific signals elicit specific responses remains elusive. To dissect different modes of ERK1/2 regulation in the nucleus and on chromatin, I took a multifaceted approach to study nuclear ERK1/2 signaling. First, I investigated the direct DNA binding activity of ERK2 in response to phosphorylation and mutation of key residues based on characterization of ERK2 phosphorylated on T188, a residue proximal to the classically defined phosphorylation activation loop sites. To test the possibility that alternate phosphorylation inputs affect ERK2 in ways other than its kinase activity, I utilized phospho-mimetic mutants to assay oligonucleotide binding. I demonstrated that phosphorylation on the activation loop enhances ERK2 association with DNA and raised the notion that this understudied property of ERK2 dictates its interactions with DNA as well as substrates in the nucleus. Second, I investigated the regulatory relationship between ERK1/2 and CXXC finger protein 1 (Cfp1). Cfp1 is an epigenetic regulator that interacts with two major chromatin modifying complexes, and my studies clarified previously collected data to demonstrate that ERK2 and Cfp1 co-regulate target genes in a signal-dependent manner. Lastly, I sought to identify the extent of ERK1 and ERK2 interactions in the nucleus. To this end, I generated tools for a proximity biotinylation strategy that will not only identify transient and stable nuclear interactions of ERK1/2, but also address the long-standing question of whether ERK1 and ERK2 perform divergent functions. Through these approaches, I identified important areas of research to improve the current understanding of the nuclear behaviors of ERK1/2 signaling and discovered that ERK1/2 regulate gene expression through multiple modes of interaction with chromatin.
Peptide Nucleic Acid (PNA) Hybridization to Nucleic Acid Targets
(2004-12-15) Nulf, Christopher J.; Corey, David R.
Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization to nucleic acid targets. The simple premise of Watson-Crick base-pairing presents PNAs with a number of diverse applications ranging fromnanotechnology to antisense therapeutics. I studied the synthesis and characterization of novel tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson-Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. I observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate binding of two inward projecting strands. These results suggested that bisPNA molecules can be used for self-assembling DNA nanostructures. The Hepatitis C Virus (HCV) RNA genome contains a conserved tertiary structure known as the internal ribosomal entry site (IRES) necessary for cap-independent translation. I tested the hypothesis that antisense peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers can bind IRES sequences and block translation. Using a lipid-mediated approach to introduce antisense PNAs and LNAs into cells, my data suggested that PNAs and LNAs could inhibit HCV IRES-dependent translation. PNA or LNA oligomers targeting different regions of the HCV IRES demonstrated a sequencespecific dose-response inhibition of translation with EC50 values of 50-150 nM. IRESdirected inhibition of gene expression widens the range of mechanisms for antisense inhibition by PNAs and LNAs and may provide further therapeutic lead compounds for the treatment of HCV. It is important to compare and contrast the biological activities of PNAs against other nucleotide analogs. Presented herein are collaborations involving comparisons of my PNAs against siRNA, 2'-O-methoxyethyl RNA, and morpholinos. Antisense PNAs were demonstrated to cause isoform-specific inhibition of protein expression of Caveolin. imilarly, PNAs demonstrated the ability to re-direct splicing activity of Insulin Receptor a-Subunit pre-mRNA. Also, antisense PNAs targeting the Chordino gene demonstrated "knock-down" morphologies similar to "knock-out" mutants in developing Zebrafish embryos. Collectively, these results suggest that PNAs are comparable in function to other oligonucleotide analogs and across many experimental platforms.
Preclinical Studies of Telomerase Inhibitor Imetelstat in Non-small Cell Lung Cancer
(2013-04-18) Frink, Robin Elizabeth; Shay, Jerry W.; Minna, John D.; Brekken, Rolf A.; White, Michael A.
Telomerase is expressed in ~90% of all cancers but is not expressed in most somatic cells making it an attractive target for cancer therapy. Telomerase has two essential components, a reverse transcriptase (hTERT) and an RNA template (hTR or hTERC). The RNA template is used by the reverse transcriptase to add the TTAGGG hexameric repeats to elongate telomeres and compensate for the loss of telomeres each cell division caused by the end replication problem. Imetelstat is an oligonucleotide designed to bind the hTR telomerase template component and inhibit telomerase leading to progressive telomere shortening associated senescence or cell death. The work described here examined the efficacy of imetelstat in a panel of non-small cell lung cancer (NSCLC) cell lines. Imetelstat was tested in a short-term liquid colony formation assay, a 5-day drug response assay, and long-term continuous treatment in vitro and in vivo. The panel of over 70 NSCLC cell lines used for this study ranged from 1.5 kb to 20 kb in average telomere length as well as a wide range in telomerase activity, growth rate, NSCLC subtype, and oncogenotype providing a broad basis for comparison of response to imetelstat. All cell lines tested showed inhibition to telomerase with imetelstat treatment. In liquid colony formation, a wide range of response to 3 uM imetelstat was seen. Colony formation inhibition ranged from 96% inhibition in HCC44 to H441 which shows a greater than 2-fold increase in colony forming ability in the presence of imetelstat, though not statistically significant. 1 uM imetelstat was given long-term in 8 different cell lines and telomerase inhibition and telomere shortening was observed in all cases. Continuous treatment led to a reduction in growth rate and eventual cell death in all but two cell lines and imetelstat response time varied among the cell lines based on initial telomere length and growth rate. Calu-3 had the fastest response time (11 days or as few as 2 population doublings) to see a change in growth rate and 32 population doublings for cell death in all cells. Calu-3, H1648 and HCC827 all showed reduced growth rate in the presence of imetelstat in vivo as well. Imetelstat inhibits telomerase, shortens telomeres and leads to cell death in many NSCLC cell lines both in vitro and in vivo supporting the idea of telomerase inhibition for the treatment of lung cancers.
Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells
(2009-01-08) Beane, Randall L.; Corey, David R.
Aberrant gene expression can lead to multiple disease-states that can be difficult or impossible to treat using traditional small-molecule medications. An alternative approach to treating such diseases is oligonucleotide-based therapeutics, which are theoretically capable of treating or curing genetic diseases, infections, and abnormalities. Oligonucleotide-based molecules targeted to DNA are referred to as antigene agents. These molecules can silence or activate gene transcription of alleles and have many potential medical applications. However, the growth of antigene technologies has been slow despite broad therapeutic potential and unique molecular applications. Through the development of chemical modifications, oligonucleotide-based molecules are actively being improved and refined. Chemical modifications can alter the cellular uptake, toxicity, biodistribution, and plasma retention of oligonucleotides. My research goal was to further the field of synthetic antigene oligonucleotides. To do this, I targeted endogenous genes in human cancer cell lines with chemically-modified oligonucleotides, including MOEs, PNAs, ENAs, and LNAs. I established that LNAs were robust antigene agents capable of inhibiting transcription under multiple conditions. Specifically, I established that mixed-base antigene agents physically associate with the hPR-B promoter and decrease the occupancy of RNA polymerase II on the hAR and hPR genes inside human cells. Furthermore, my research indicates that antigene LNAs function in an orientation-dependent manner and that functional LNAs must target the template strand of DNA to have appreciable potency. This body of work comprised the first extensive study of a mixed-base antigene oligonucleotide in multiple human cell lines and provides the first evidence that mixed-base antigene agents can physically associate with chromosomal DNA and inhibit transcription of endogenous mammalian genes inside human cells. Collectively, my data suggest that antigene LNAs are a potent and general strategy for silencing gene expression, and that antigene LNAs also have potential therapeutic applications and possible utility in modern functional genomics.
[Southwestern News]
(1996-11-25) Steeves, Susan A.