Cindy Meyer

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Rockefeller University


Scopus Publications

Scopus Publications

  • Publisher Correction: Expanding the binding specificity for RNA recognition by a PUF domain (Nature Communications, (2021), 12, 1, (5107), 10.1038/s41467-021-25433-6)
    Wei Zhou, Daniel Melamed, Gabor Banyai, Cindy Meyer, Thomas Tuschl, Marvin Wickens, Junyue Cao, and Stanley Fields

    Nature Communications, eISSN: 20411723, Published: December 2022 Springer Science and Business Media LLC

  • Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture
    Xuanting Wang, Carolina Q. Sacramento, Steffen Jockusch, Otávio Augusto Chaves, Chuanjuan Tao, Natalia Fintelman-Rodrigues, Minchen Chien, Jairo R. Temerozo, Xiaoxu Li, Shiv Kumar, Wei Xie, Dinshaw J. Patel, Cindy Meyer, Aitor Garzia, Thomas Tuschl, Patrícia T. Bozza, James J. Russo, Thiago Moreno L. Souza, and Jingyue Ju

    Communications Biology, eISSN: 23993642, Published: December 2022 Springer Science and Business Media LLC
    AbstractSARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

  • Expanding the binding specificity for RNA recognition by a PUF domain
    Wei Zhou, Daniel Melamed, Gabor Banyai, Cindy Meyer, Thomas Tuschl, Marvin Wickens, Junyue Cao, and Stanley Fields

    Nature Communications, eISSN: 20411723, Published: 1 December 2021 Springer Science and Business Media LLC
    AbstractThe ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

  • The E3 ubiquitin ligase RNF10 modifies 40S ribosomal subunits of ribosomes compromised in translation
    Aitor Garzia, Cindy Meyer, and Thomas Tuschl

    Cell Reports, eISSN: 22111247, Published: 3 August 2021 Elsevier BV
    Reversible monoubiquitination of small subunit ribosomal proteins RPS2/uS5 and RPS3/uS3 has been noted to occur on ribosomes involved in ZNF598-dependent mRNA surveillance. Subsequent deubiquitination of RPS2 and RPS3 by USP10 is critical for recycling of stalled ribosomes in a process known as ribosome-associated quality control. Here, we identify and characterize the RPS2- and RPS3-specific E3 ligase Really Interesting New Gene (RING) finger protein 10 (RNF10) and its role in translation. Overexpression of RNF10 increases 40S ribosomal subunit degradation similarly to the knockout of USP10. Although a substantial fraction of RNF10-mediated RPS2 and RPS3 monoubiquitination results from ZNF598-dependent sensing of collided ribosomes, ZNF598-independent impairment of translation initiation and elongation also contributes to RPS2 and RPS3 monoubiquitination. RNF10 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies crosslinked mRNAs, tRNAs, and 18S rRNAs, indicating recruitment of RNF10 to ribosomes stalled in translation. These impeded ribosomes are tagged by ubiquitin at their 40S subunit for subsequent programmed degradation unless rescued by USP10.

  • The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation
    Cindy Meyer, Aitor Garzia, Pavel Morozov, Henrik Molina, and Thomas Tuschl

    Molecular Cell, ISSN: 10972765, eISSN: 10974164, Pages: 1193-1205.e5, Published: 19 March 2020 Elsevier BV
    Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.

  • The RNA-Binding Protein A1CF Regulates Hepatic Fructose and Glycerol Metabolism via Alternative RNA Splicing
    Kostas C. Nikolaou, Hasan Vatandaslar, Cindy Meyer, Marc W. Schmid, Thomas Tuschl, and Markus Stoffel

    Cell Reports, eISSN: 22111247, Pages: 283-300.e8, Published: 8 October 2019 Elsevier BV
    The regulation of hepatic gene expression has been extensively studied at the transcriptional level; however, the control of metabolism through posttranscriptional gene regulation by RNA-binding proteins in physiological and disease states is less understood. Here, we report a major role for the hormone-sensitive RNA-binding protein (RBP) APOBEC1 complementation factor (A1CF) in the generation of hepatocyte-specific and alternatively spliced transcripts. Among these transcripts are isoforms for the dominant and high-affinity fructose-metabolizing ketohexokinase C and glycerol kinase, two key metabolic enzymes that are linked to hepatic gluconeogenesis and found to be markedly reduced upon hepatic ablation of A1cf. Consequently, mice lacking A1CF exhibit improved glucose tolerance and are protected from fructose-induced hyperglycemia, hepatic steatosis, and development of obesity. Our results identify a previously unreported function of A1CF as a regulator of alternative splicing of a subset of genes influencing hepatic glucose production through fructose and glycerol metabolism.

  • The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research
    Saumya Das, K. Mark Ansel, Markus Bitzer, Xandra O. Breakefield, Alain Charest, David J. Galas, Mark B. Gerstein, Mihir Gupta, Aleksandar Milosavljevic, Michael T. McManus, Tushar Patel, Robert L. Raffai, Joel Rozowsky, Matthew E. Roth, Julie A. Saugstad, Kendall Van Keuren-Jensen, Alissa M. Weaver, Louise C. Laurent, Asim B. Abdel-Mageed, Catherine Adamidi, P. David Adelson, Kemal M. Akat, Eric Alsop, K. Mark Ansel, Jorge Arango, Neil Aronin, Seda Kilinc Avsaroglu, Azadeh Azizian, Leonora Balaj, Iddo Z. Ben-Dov, Karl Bertram, Markus Bitzer, Robert Blelloch, Kimberly A. Bogardus, Xandra Owens Breakefield, George A. Calin, Bob S. Carter, Al Charest, Clark C. Chen, Tanuja Chitnis, Robert J. Coffey, Amanda Courtright-Lim, Saumya Das, Amrita Datta, Peter DeHoff, Thomas G. Diacovo, David J. Erle, Alton Etheridge, Marc Ferrer, Jeffrey L. Franklin, Jane E. Freedman, David J. Galas, Timur Galeev, Roopali Gandhi, Aitor Garcia, Mark Bender Gerstein, Vikas Ghai, Ionita Calin Ghiran, Maria D. Giraldez, Andrei Goga, Tasos Gogakos, Beatrice Goilav, Stephen J. Gould, Peixuan Guo, Mihir Gupta, Fred Hochberg, Bo Huang, Matt Huentelman, Craig Hunter, Elizabeth Hutchins, Andrew R. Jackson, M. Yashar S. Kalani, Pinar Kanlikilicer, Reka Agnes Karaszti, Kendall Van Keuren-Jensen, Anastasia Khvorova, Yong Kim, Hogyoung Kim, Taek Kyun Kim, Robert Kitchen, Richard P. Kraig, Anna M. Krichevsky, Raymond Y. Kwong, Louise C. Laurent, Minyoung Lee, Noelle L’Etoile, Shawn E. Levy, Feng Li, Jenny Li, Xin Li, Gabriel Lopez-Berestein, Rocco Lucero, Bogdan Mateescu, A.C. Matin, Klaas E.A. Max, Michael T. McManus, Thorsten R. Mempel, Cindy Meyer, Aleksandar Milosavljevic, Debasis Mondal, Kenneth Jay Mukamal, Oscar D. Murillo, Thangamani Muthukumar, Deborah A. Nickerson, Christopher J. O’Donnell, Dinshaw J. Patel, Tushar Patel, James G. Patton, Anu Paul, Elaine R. Peskind, Mitch A. Phelps, Chaim Putterman, Peter J. Quesenberry, Joseph F. Quinn, Robert L. Raffai, Saritha Ranabothu, Shannon Jiang Rao, Cristian Rodriguez-Aguayo, Anthony Rosenzweig, Matthew E. Roth, Joel Rozowsky, Marc S. Sabatine, Nikita A. Sakhanenko, Julie Anne Saugstad, Thomas D. Schmittgen, Neethu Shah, Ravi Shah, Kerby Shedden, Jian Shi, Anil K. Sood, Anuoluwapo Sopeyin, Ryan M. Spengler, Robert Spetzler, Srimeenakshi Srinivasan, Sai Lakshmi Subramanian, Manikkam Suthanthiran, Kahraman Tanriverdi, Yun Teng, Muneesh Tewari, William Thistlethwaite, Thomas Tuschl, Karolina Kaczor Urbanowicz, Kasey C. Vickers, Olivier Voinnet, Kai Wang, Alissa M. Weaver, Zhiyun Wei, Howard L. Weiner, Zachary R. Weiss, Zev Williams, David T.W. Wong, Prescott G. Woodruff, Xinshu Xiao, Irene K. Yan, Ashish Yeri, Bing Zhang, and Huang-Ge Zhang
    ISSN: 00928674, eISSN: 10974172, Volume: 177, Pages: 231-242, Published: 4 April 2019 Elsevier BV
    The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

  • The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression
    Cindy Meyer, Aitor Garzia, Michael Mazzola, Stefanie Gerstberger, Henrik Molina, and Thomas Tuschl

    Molecular Cell, ISSN: 10972765, eISSN: 10974164, Pages: 622-635.e6, Published: 15 February 2018 Elsevier BV
    TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

  • PAR-CLIP for discovering target sites of RNA-binding proteins
    Aitor Garzia, Pavel Morozov, Marcin Sajek, Cindy Meyer, and Thomas Tuschl

    Methods in Molecular Biology, ISSN: 10643745, Volume: 1720, Pages: 55-75, Published: 2018 Springer New York
    RNA-binding proteins (RBPs) establish posttranscriptional gene regulation (PTGR) by coordinating the maturation, editing, transport, stability, and translation of cellular RNAs. A variety of experimental approaches have been developed to characterize the RNAs associated with RBPs in vitro as well as in vivo. Our laboratory developed Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP), which in combination with next-generation sequencing enables the identification of RNA targets of RBPs at a nucleotide-level resolution. Here we present an updated and condensed step-by-step PAR-CLIP protocol followed by the description of our RNA-seq data analysis pipeline.

  • The Conserved RNA Exonuclease Rexo5 Is Required for 3′ End Maturation of 28S rRNA, 5S rRNA, and snoRNAs
    Stefanie Gerstberger, Cindy Meyer, Sigi Benjamin-Hong, Joe Rodriguez, Daniel Briskin, Claudia Bognanni, Kimberly Bogardus, Hermann Steller, and Thomas Tuschl

    Cell Reports, eISSN: 22111247, Pages: 758-772, Published: 17 October 2017 Elsevier BV
    Non-coding RNA biogenesis in higher eukaryotes has not been fully characterized. Here, we studied the Drosophila melanogaster Rexo5 (CG8368) protein, a metazoan-specific member of the DEDDh 3'-5' single-stranded RNA exonucleases, by genetic, biochemical, and RNA-sequencing approaches. Rexo5 is required for small nucleolar RNA (snoRNA) and rRNA biogenesis and is essential in D. melanogaster. Loss-of-function mutants accumulate improperly 3' end-trimmed 28S rRNA, 5S rRNA, and snoRNA precursors in vivo. Rexo5 is ubiquitously expressed at low levels in somatic metazoan cells but extremely elevated in male and female germ cells. Loss of Rexo5 leads to increased nucleolar size, genomic instability, defective ribosome subunit export, and larval death. Loss of germline expression compromises gonadal growth and meiotic entry during germline development.

  • Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP
    Tasos Gogakos, Miguel Brown, Aitor Garzia, Cindy Meyer, Markus Hafner, and Thomas Tuschl

    Cell Reports, eISSN: 22111247, Pages: 1463-1475, Published: 8 August 2017 Elsevier BV
    The participation of tRNAs in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes and curation of tRNA sequences. This has been challenging because RNA secondary structure, nucleotide modifications, and tRNA gene multiplicity complicate sequencing and mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with photoactivatable crosslinking and immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report nucleotide modification sites and their order of introduction, and we identify tRNA leaders, trailers, and introns. By using complementary sequencing-based methodologies, we present a human tRNA atlas and determine expression levels of mature and processing intermediates of tRNAs in human cells.

  • The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs
    Aitor Garzia, Seyed Mehdi Jafarnejad, Cindy Meyer, Clément Chapat, Tasos Gogakos, Pavel Morozov, Mehdi Amiri, Maayan Shapiro, Henrik Molina, Thomas Tuschl, and Nahum Sonenberg

    Nature Communications, eISSN: 20411723, Published: 7 July 2017 Springer Science and Business Media LLC
    Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

  • Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins
    Aitor Garzia, Cindy Meyer, Pavel Morozov, Marcin Sajek, and Thomas Tuschl

    Methods, ISSN: 10462023, eISSN: 10959130, Volume: 118-119, Pages: 24-40, Published: 15 April 2017 Elsevier BV
    Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.

  • Simultaneous detection of the subcellular localization of RNAs and proteins in cultured cells by combined multicolor RNA-FISH and IF
    Cindy Meyer, Aitor Garzia, and Thomas Tuschl

    Methods, ISSN: 10462023, eISSN: 10959130, Volume: 118-119, Pages: 101-110, Published: 15 April 2017 Elsevier BV
    Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions.

  • DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs
    Masashi Yamaji, Miki Jishage, Cindy Meyer, Hemant Suryawanshi, Evan Der, Misaki Yamaji, Aitor Garzia, Pavel Morozov, Sudhir Manickavel, Hannah L. McFarland, Robert G. Roeder, Markus Hafner, and Thomas Tuschl

    Nature, ISSN: 00280836, eISSN: 14764687, Volume: 543, Issue: 7646, Pages: 568-572, Published: 23 March 2017 Springer Science and Business Media LLC
    The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3′ untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4–NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFβ superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

  • RAID3 - An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity
    Florian Mittelberger, Cindy Meyer, Georg H Waetzig, Martin Zacharias, Erica Valentini, Dmitri I Svergun, Katharina Berg, Inken Lorenzen, Joachim Grötzinger, Stefan Rose-John, and Ulrich Hahn

    RNA Biology, ISSN: 15476286, eISSN: 15558584, Pages: 1043-1053, Published: 2015 Informa UK Limited
    Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule. Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2′-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

  • SDA, a DNA aptamer inhibiting E- And P-Selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation
    Rassa Faryammanesh, Tobias Lange, Eileen Magbanua, Sina Haas, Cindy Meyer, Daniel Wicklein, Udo Schumacher, and Ulrich Hahn

    PLoS ONE, eISSN: 19326203, Published: 3 April 2014 Public Library of Science (PLoS)
    Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

  • Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets
    T. A. Farazi, C. S. Leonhardt, N. Mukherjee, A. Mihailovic, S. Li, K. E. A. Max, C. Meyer, M. Yamaji, P. Cekan, N. C. Jacobs, S. Gerstberger, C. Bognanni, E. Larsson, U. Ohler, and T. Tuschl
    ISSN: 13558382, eISSN: 14699001, Pages: 1090-1102, Published: July 2014 Cold Spring Harbor Laboratory
    Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

  • Stabilized interleukin-6 receptor binding RNA aptamers
    Cindy Meyer, Katharina Berg, Katja Eydeler-Haeder, Inken Lorenzen, Joachim Grötzinger, Stefan Rose-John, and Ulrich Hahn

    RNA Biology, ISSN: 15476286, eISSN: 15558584, Pages: 57-65, Published: January 2014 Informa UK Limited
    Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in the progression of various inflammatory diseases, such as rheumatoid arthritis and certain cancers; for example, multiple myeloma or hepatocellular carcinoma. To interfere with IL-6-dependent diseases, targeting IL-6 receptor (IL-6R)-presenting tumor cells using aptamers might be a valuable strategy to broaden established IL-6- or IL-6R-directed treatment regimens. Recently, we reported on the in vitro selection of RNA aptamers binding to the human IL-6 receptor (IL-6R) with nanomolar affinity. One aptamer, namely AIR-3A, was 19 nt in size and able to deliver bulky cargos into IL-6R-presenting cells. As AIR-3A is a natural RNA molecule, its use for in vivo applications might be limited due to its susceptibility to ubiquitous ribonucleases. Aiming at more robust RNA aptamers targeting IL-6R, we now report on the generation of stabilized RNA aptamers for potential in vivo applications. The new 2'-F-modified RNA aptamers bind to IL-6R via its extracellular portion with low nanomolar affinity comparable to the previously identified unmodified counterpart. Aptamers do not interfere with the IL-6 receptor complex formation. The work described here represents one further step to potentially apply stabilized IL-6R-binding RNA aptamers in IL-6R-connected diseases, like multiple myeloma and hepatocellular carcinoma.

  • Chlorin e6 conjugated interleukin-6 receptor aptamers selectively kill target cells upon irradiation
    Sven Kruspe, Cindy Meyer, and Ulrich Hahn

    Molecular Therapy - Nucleic Acids, eISSN: 21622531, Published: 2014 Elsevier BV
    Photodynamic therapy (PDT) uses the therapeutic properties of light in combination with certain chemicals, called photosensitizers, to successfully treat brain, breast, prostate, and skin cancers. To improve PDT, current research focuses on the development of photosensitizers to specifically target cancer cells. In the past few years, aptamers have been developed to directly deliver cargo molecules into target cells. We conjugated the photosensitizer chlorin e6 (ce6) with a human interleukin-6 receptor (IL-6R) binding RNA aptamer, AIR-3A yielding AIR-3A-ce6 for application in high efficient PDT. AIR-3A-ce6 was rapidly and specifically internalized by IL-6R presenting (IL-6R+) cells. Upon light irradiation, targeted cells were selectively killed, while free ce6 did not show any toxic effect. Cells lacking the IL-6R were also not affected by AIR-3A-ce6. With this approach, we improved the target specificity of ce6-mediated PDT. In the future, other tumor-specific aptamers might be used to selectively localize photosensitizers into cells of interest and improve the efficacy and specificity of PDT in cancer and other diseases.

  • Rna aptamer design
    Cindy Meyer, Ulrich Hahn, and Andrew E. Torda

    De novo Molecular Design, Pages: 519-542, Published: 11 October 2013 Wiley-VCH Verlag GmbH & Co. KGaA

  • D(GGGT)<inf>4</inf> and r(GGGU)<inf>4</inf> are both HIV-1 inhibitors and interleukin-6 receptor aptamers
    Eileen Magbanua, Tijana Zivkovic, Björn Hansen, Niklas Beschorner, Cindy Meyer, Inken Lorenzen, Joachim Grötzinger, Joachim Hauber, Andrew E. Torda, Günter Mayer, Stefan Rose-John, and Ulrich Hahn

    RNA Biology, ISSN: 15476286, eISSN: 15558584, Pages: 216-227, Published: February 2013 Informa UK Limited
    Aptamers are oligonucleotides that bind targets with high specificity and affinity. They have become important tools for biosensing, target detection, drug delivery and therapy. We selected the quadruplex-forming 16-mer DNA aptamer AID-1 [d(GGGT)4] with affinity for the interleukin-6 receptor (IL-6R) and identified single nucleotide variants that showed no significant loss of binding ability. The RNA counterpart of AID-1 [r(GGGU)4] also bound IL-6R as quadruplex structure. AID-1 is identical to the well-known HIV inhibitor T30923, which inhibits both HIV infection and HIV-1 integrase. We also demonstrated that IL-6R specific RNA aptamers not only bind HIV-1 integrase and inhibit its 3′ processing activity in vitro, but also are capable of preventing HIV de novo infection with the same efficacy as the established inhibitor T30175. All these aptamer target interactions are highly dependent on formation of quadruplex structure.

  • Human α <inf>2</inf>-macroglobulin-another variation on the Venus flytrap
    Cindy Meyer, Winfried Hinrichs, and Ulrich Hahn

    Angewandte Chemie - International Edition, ISSN: 14337851, eISSN: 15213773, Pages: 5045-5047, Published: 21 May 2012 Wiley

  • Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells
    Cindy Meyer, Katja Eydeler, Eileen Magbanua, Tijana Zivkovic, Nicolas Piganeau, Inken Lorenzen, Joachim Grötzinger, Günter Mayer, Stefan Rose-John, and Ulrich Hahn

    RNA Biology, ISSN: 15476286, eISSN: 15558584, Pages: 67-80, Published: January 2012 Informa UK Limited
    Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.

  • Cell-specific aptamers as emerging therapeutics
    Cindy Meyer, Ulrich Hahn, and Andrea Rentmeister

    Journal of Nucleic Acids, ISSN: 20900201, eISSN: 2090021X, Volume: 2011, Published: 2011 Hindawi Limited
    Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by anin vitroprocess named SELEX (systematic evolution of ligands by exponential enrichment). Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.