Publications by authors named "James J Russo"

43 Publications

Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics.

bioRxiv 2021 Jul 21. Epub 2021 Jul 21.

SARS-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.
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http://dx.doi.org/10.1101/2021.07.21.453274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8312893PMC
July 2021

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

J Antimicrob Chemother 2021 06;76(7):1874-1885

Instituto D'Or de Pesquisa e Ensino, Rio de Janeiro, RJ, Brazil.

Background: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.

Methods: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.

Results: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.

Conclusions: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.
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http://dx.doi.org/10.1093/jac/dkab072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083231PMC
June 2021

Nucleotide analogues as inhibitors of SARS-CoV Polymerase.

Pharmacol Res Perspect 2020 12;8(6):e00674

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, New York, USA.

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.
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http://dx.doi.org/10.1002/prp2.674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596664PMC
December 2020

Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir.

Sci Rep 2020 10 6;10(1):16577. Epub 2020 Oct 6.

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY, 10027, USA.

SARS-CoV-2 is responsible for COVID-19, resulting in the largest pandemic in over a hundred years. After examining the molecular structures and activities of hepatitis C viral inhibitors and comparing hepatitis C virus and coronavirus replication, we previously postulated that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) might inhibit SARS-CoV-2. We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RNA-dependent RNA polymerases (RdRps), serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. Other investigators have since demonstrated the ability of Sofosbuvir to inhibit SARS-CoV-2 replication in lung and brain cells; additionally, COVID-19 clinical trials with EPCLUSA and with Sofosbuvir plus Daclatasvir have been initiated in several countries. SARS-CoV-2 has an exonuclease-based proofreader to maintain the viral genome integrity. Any effective antiviral targeting the SARS-CoV-2 RdRp must display a certain level of resistance to this proofreading activity. We report here that Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher extent than RNA terminated by Remdesivir, another drug being used as a COVID-19 therapeutic. These results offer a molecular basis supporting the current use of Sofosbuvir in combination with other drugs in COVID-19 clinical trials.
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http://dx.doi.org/10.1038/s41598-020-73641-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538426PMC
October 2020

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19.

J Proteome Res 2020 11 5;19(11):4690-4697. Epub 2020 Aug 5.

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.
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http://dx.doi.org/10.1021/acs.jproteome.0c00392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640960PMC
November 2020

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19.

Antiviral Res 2020 08 18;180:104857. Epub 2020 Jun 18.

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY, 10027, USA; Department of Chemical Engineering, Columbia University, New York, NY, 10027, USA; Department of Pharmacology, Columbia University, New York, NY, 10027, USA. Electronic address:

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.
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http://dx.doi.org/10.1016/j.antiviral.2020.104857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299870PMC
August 2020

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase.

bioRxiv 2020 Mar 20. Epub 2020 Mar 20.

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). Here, using polymerase extension experiments, we have demonstrated that the active triphosphate form of Sofosbuvir (a key component of the FDA approved hepatitis C drug EPCLUSA), is incorporated by SARS-CoV-2 RdRp, and blocks further incorporation. Using the same molecular insight, we selected the active triphosphate forms of three other anti-viral agents, Alovudine, AZT (an FDA approved HIV/AIDS drug) and Tenofovir alafenamide (TAF, an FDA approved drug for HIV and hepatitis B) for evaluation as inhibitors of SARS-CoV-2 RdRp. We demonstrated the ability of these three viral polymerase inhibitors, 3'-fluoro-3'-deoxythymidine triphosphate, 3'-azido-3'-deoxythymidine triphosphate and Tenofovir diphosphate (the active triphosphate forms of Alovudine, AZT and TAF, respectively) to be incorporated by SARS-CoV-2 RdRp, where they also terminate further polymerase extension. These results offer a strong molecular basis for these nucleotide analogues to be evaluated as potential therapeutics for COVID-19.
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http://dx.doi.org/10.1101/2020.03.18.997585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239050PMC
March 2020

Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications.

ACS Chem Biol 2020 06 19;15(6):1464-1472. Epub 2020 May 19.

Department of Biological and Chemical Sciences, New York Institute of Technology, New York, New York 10023, United States.

Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNA without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNA was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2'-O-methylated G (Gm) in the wobble anticodon position as well as an -dimethylguanosine (mG), a 7-methylguanosine (mG), a 1-methyladenosine (mA), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNA (75 nt with a 3'-CC tail (80% abundance) and 74 nt with a 3'-C tail (3% abundance)) were identified in addition to the full-length 3'-CCA-tailed tRNA (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNA variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.
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http://dx.doi.org/10.1021/acschembio.0c00119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902080PMC
June 2020

Photochemical conversion of a cytidine derivative to a thymidine analog via [2+2]-cycloaddition.

Photochem Photobiol Sci 2018 Aug;17(8):1049-1055

Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027, USA.

Epigenetic information is encoded in the mammalian genome in the form of cytosines methylated at the 5 position. Cytosine methylation has multiple biological effects, but our understanding of these effects has lagged because extant methods for mapping methylation sites genome-wide have severe shortcomings. For instance, the gold standard bisulfite sequencing approach suffers from the use of harsh reaction conditions resulting in DNA cleavage and incomplete conversion of unmethylated cytosine to uracil. We report here on a new photochemical method in which a DNA (cytosine-5)-methyltransferase can be used to covalently attach reactive functionalities which upon irradiation at ∼350 nm initiate photoinduced intramolecular reactions that convert modified C to T analogues. We synthesized a model compound, a cinnamyl ether-containing cytidine derivative, and demonstrated its conversion to a thymidine analogue using mild conditions and a DNA-compatible wavelength (∼350 nm), enabled by the use of a triplet sensitizer, thioxanthone. Transfer of a cinnamyl ether or comparable reactive functionality from an AdoMet analog to cytosine followed by the use of this photoconversion method would require only small amounts of DNA and allow complete methylation profiling on both long and short read sequencing platforms.
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http://dx.doi.org/10.1039/c8pp00161hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082695PMC
August 2018

Saturation mutagenesis reveals manifold determinants of exon definition.

Genome Res 2018 01 14;28(1):11-24. Epub 2017 Dec 14.

Department of Biological Sciences, Columbia University, New York, New York 10027, USA.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.
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http://dx.doi.org/10.1101/gr.219683.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749175PMC
January 2018

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array.

Proc Natl Acad Sci U S A 2016 11 11;113(44):E6749-E6756. Epub 2016 Oct 11.

Department of Genetics, Harvard Medical School, Boston, MA 02115;

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin-polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.
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http://dx.doi.org/10.1073/pnas.1608271113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098637PMC
November 2016

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

Proc Natl Acad Sci U S A 2016 May 18;113(19):5233-8. Epub 2016 Apr 18.

Genia Technologies, Santa Clara, CA 95050;

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.
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http://dx.doi.org/10.1073/pnas.1601782113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868432PMC
May 2016

Identification of miR-215 mediated targets/pathways via translational immunoprecipitation expression analysis (TrIP-chip).

Oncotarget 2015 Sep;6(27):24463-73

Department of Pathology, Stony Brook University, School of Medicine, Stony Brook, NY, USA.

Steady state mRNA expression profiling can identify the majority of miRNA targets. However, some translationally repressed miRNA targets are missed and thus not considered for functional validation. Therefore, analysis of mRNA translation can enhance miRNA target identification for functional studies. We have applied a unique approach to identify miRNA targets in a small number of cells. Actively translating mRNAs are associated with polyribosomes and newly synthesized peptide chains are associated with molecular chaperones such as HSP70s. Affinity capture beads were used to capture HSP70 chaperones associated with polyribosome complexes. The isolated actively translating mRNAs were used for high throughput expression profiling analysis. miR-215 is an important miRNA in colorectal cancer and loss of miR-215 is significantly associated with prognosis of this disease. miR-215 suppresses the expression of several key targets. We utilized the affinity capture approach to isolate miR-215 mediated mRNA target transcripts. This approach provides a unique way to identify targets regulated by non-coding RNAs and RNA binding proteins from a small number of cells.
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http://dx.doi.org/10.18632/oncotarget.4425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695198PMC
September 2015

DNA sequencing by synthesis using 3'-azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection.

RSC Adv 2014 Jan;4(90):49342-49346

Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Chemical Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Pharmacology, Columbia University, 3000 Broadway, New York, NY 10027, USA.

As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report here an approach using an azido moiety (N) that has an intense, narrow and unique Raman shift at 2125 cm, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3'-azidomethyl nucleotide reversible terminators (3'-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm as a result of polymerase extension by a single 3'-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3'-azidomethyl-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.
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http://dx.doi.org/10.1039/C4RA08398ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226440PMC
January 2014

A Microfluidic Device for Multiplex Single-Nucleotide Polymorphism Genotyping.

RSC Adv 2014 Jan 7;4(9):4269-4277. Epub 2013 Nov 7.

Department of Mechanical Engineering, Columbia University, New York, NY, 10027.

Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics.
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http://dx.doi.org/10.1039/c3ra44091eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651459PMC
January 2014

A strategy to capture and characterize the synaptic transcriptome.

Proc Natl Acad Sci U S A 2013 Apr 15;110(18):7464-9. Epub 2013 Apr 15.

Department of Neuroscience and Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA.

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence.
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http://dx.doi.org/10.1073/pnas.1304422110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645558PMC
April 2013

PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

Sci Rep 2012 21;2:684. Epub 2012 Sep 21.

Center for Genome Technology & Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027, USA.

We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.
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http://dx.doi.org/10.1038/srep00684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448304PMC
February 2013

Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides.

Anal Biochem 2012 Aug 10;427(2):202-10. Epub 2012 May 10.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.
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http://dx.doi.org/10.1016/j.ab.2012.05.001DOI Listing
August 2012

Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Anal Biochem 2012 Aug 25;427(2):193-201. Epub 2012 Apr 25.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based methods have been widely explored for DNA sequencing. We report here the design, synthesis, and evaluation of a novel set of chemically cleavable biotinylated dideoxynucleotides, ddNTPs-N₃-biotin, for the DNA polymerase extension reaction and its application in DNA sequencing by mass spectrometry (MS). These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute to large mass differences among the nucleotides. We demonstrate that these modified nucleotides are efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides is captured by streptavidin-coated beads and efficiently released using tris(2-carboxyethyl)phosphine in aqueous solution, which is compatible with DNA and downstream procedures. We performed Sanger sequencing reactions using these nucleotides to generate DNA fragments for MALDI-TOF MS analysis. Both synthetic DNA and polymerase chain reaction (PCR) products were accurately decoded, and a read length of approximately 37 bases was achieved using these nucleotides in MS sequencing.
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http://dx.doi.org/10.1016/j.ab.2012.04.021DOI Listing
August 2012

Quantitative evaluation of all hexamers as exonic splicing elements.

Genome Res 2011 Aug 9;21(8):1360-74. Epub 2011 Jun 9.

Department of Biological Sciences, Columbia University, New York, New York 10027, USA.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 3' splice sites tend to be single stranded, and effective 5' splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.
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http://dx.doi.org/10.1101/gr.119628.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149502PMC
August 2011

Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip).

Nucleic Acids Res 2010 May 31;38(9):e104. Epub 2010 Jan 31.

Mitchell Cancer Institute, Mobile, AL 36688, USA.

We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
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http://dx.doi.org/10.1093/nar/gkq024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875024PMC
May 2010

Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.

Neuron 2009 Sep;63(6):803-17

Department of Neuroscience, Columbia University, New York, NY 10032, USA.

Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. To explore the role of small regulatory RNAs in learning-related synaptic plasticity, we carried out massive parallel sequencing to profile the small RNAs of Aplysia californica. We identified 170 distinct miRNAs, 13 of which were novel and specific to Aplysia. Nine miRNAs were brain enriched, and several of these were rapidly downregulated by transient exposure to serotonin, a modulatory neurotransmitter released during learning. Further characterization of the brain-enriched miRNAs revealed that miR-124, the most abundant and well-conserved brain-specific miRNA, was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB. We therefore present direct evidence that a modulatory neurotransmitter important for learning can regulate the levels of small RNAs and present a role for miR-124 in long-term plasticity of synapses in the mature nervous system.
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http://dx.doi.org/10.1016/j.neuron.2009.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875683PMC
September 2009

SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.

J Bacteriol 2009 Apr 13;191(8):2461-73. Epub 2009 Feb 13.

Department of Microbiology, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA.

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.
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http://dx.doi.org/10.1128/JB.01578-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668410PMC
April 2009

Centrotemporal sharp wave EEG trait in rolandic epilepsy maps to Elongator Protein Complex 4 (ELP4).

Eur J Hum Genet 2009 Sep 28;17(9):1171-81. Epub 2009 Jan 28.

Child Health Evaluative Sciences, The Hospital for Sick Children, Toronto, Ontario, Canada.

Rolandic epilepsy (RE) is the most common human epilepsy, affecting children between 3 and 12 years of age, boys more often than girls (3:2). Focal sharp waves in the centrotemporal area define the electroencephalographic (EEG) trait for the syndrome, are a feature of several related childhood epilepsies and are frequently observed in common developmental disorders (eg, speech dyspraxia, attention deficit hyperactivity disorder and developmental coordination disorder). Here we report the first genome-wide linkage scan in RE for the EEG trait, centrotemporal sharp waves (CTS), with genome-wide linkage of CTS to 11p13 (HLOD 4.30). Pure likelihood statistical analysis refined our linkage peak by fine mapping CTS to variants in Elongator Protein Complex 4 (ELP4) in two independent data sets; the strongest evidence was with rs986527 in intron 9 of ELP4, providing a likelihood ratio of 629:1 (P=0.0002) in favor of an association. Resequencing of ELP4 coding, flanking and promoter regions revealed no significant exonic polymorphisms. This is the first report of a gene implicated in a common focal epilepsy and the first human disease association of ELP4. ELP4 is a component of the Elongator complex, involved in transcription and tRNA modification. Elongator depletion results in the brain-specific downregulation of genes implicated in cell motility and migration. We hypothesize that a non-coding mutation in ELP4 impairs brain-specific Elongator-mediated interaction of genes implicated in brain development, resulting in susceptibility to seizures and neurodevelopmental disorders.
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http://dx.doi.org/10.1038/ejhg.2008.267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729813PMC
September 2009

ReproArray(GTS): a cDNA microarray for identification of reproduction-related genes in the giant tiger shrimp Penaeus monodon and characterization of a novel nuclear autoantigenic sperm protein (NASP) gene.

Comp Biochem Physiol Part D Genomics Proteomics 2009 Jun 10;4(2):90-9. Epub 2008 Dec 10.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Klong 1, Klong Luang, Pathumthani 12120, Thailand.

Expressed sequence tags (ESTs) were established from various tissues of the giant tiger shrimp (Penaeus monodon). To simultaneously examine expression patterns of a large number of transcripts in ovaries and testes of P. monodon, a cDNA microarray (ReproArray(GTS)) containing 4992 features amplified from cDNAs of ovary (1920) and testis (3072) EST libraries was constructed and subjected to high-throughput gene expression analysis in four different stages of ovarian development (previtellogenesis, vitellogenesis, early cortical rod and late cortical rod stages). Several transcripts were found to be differentially expressed during P. monodon ovarian development. Among many important reproduction-related genes with differential expression from microarray data, nuclear autoantigenic sperm protein (NASP) was further characterized by RACE-PCR. The full-length cDNA of P. monodon NASP (PmNASP) was 2126 bp in length containing an open reading frame (ORF) of 1812 bp corresponding to a deduced protein of 603 amino acids with 5? and 3?UTRs of 93 and 202 bp (excluding the poly A tail), respectively. Higher PmNASP transcript levels at later stages of ovarian development was consistently confirmed by quantitative real-time PCR. This study indicated that ReproArray(GTS) is effective for high-throughput screening of genes that play important roles in ovarian development of P. monodon.
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http://dx.doi.org/10.1016/j.cbd.2008.11.003DOI Listing
June 2009

Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.

Proc Natl Acad Sci U S A 2008 Jul 30;105(27):9145-50. Epub 2008 Jun 30.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.
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http://dx.doi.org/10.1073/pnas.0804023105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442126PMC
July 2008

Endothelial nitric oxide synthase gene single-nucleotide polymorphism predicts cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

J Cereb Blood Flow Metab 2008 Jun 5;28(6):1204-11. Epub 2008 Mar 5.

Department of Neurological Surgery, Columbia University, New York, New York, USA.

Vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (aSAH). Studies have shown a link between single-nucleotide polymorphisms (SNPs) in the endothelial nitric oxide synthase (eNOS) gene and the incidence of coronary spasm and aneurysms. Alterations in the eNOS T-786 SNP may lead to an increased risk of post-aSAH cerebral vasospasm. In this prospective clinical study, 77 aSAH patients provided genetic material and were followed for the occurrence of vasospasm. In multivariate logistic regression analysis, genotype was the only factor predictive of vasospasm. The odds ratio (OR) for symptomatic vasospasm in patients with one T allele was 3.3 (95% confidence interval (CI): 1.1 to 10.0, P=0.034) and 10.9 for TT. Patients with angiographic spasm were 3.6 times more likely to have a T allele (95% CI: 1.3 to 9.6, P=0.013; for TT: OR 12.6). Patients with severe vasospasm requiring endovascular therapy were more likely to have a T allele (OR 3.5, 95% CI: 1.3 to 9.5, P=0.016; for TT: OR 12.0). Patients with the T allele of the eNOS gene are more likely to have severe vasospasm. Presence of this genotype may allow the identification of individuals at high risk for post-aSAH vasospasm and lead to early treatment and improved outcome.
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http://dx.doi.org/10.1038/jcbfm.2008.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744963PMC
June 2008

Prognostic Values of microRNAs in Colorectal Cancer.

Biomark Insights 2006 ;2:113-121

The Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36688.

The functions of non-coding microRNAs (miRNAs) in tumorigenesis are just beginning to emerge. Previous studies from our laboratory have identified a number of miRNAs that were deregulated in colon cancer cell lines due to the deletion of the p53 tumor suppressor gene. In this study, the in vivo significance of some of these miRNAs was further evaluated using colorectal clinical samples. Ten miRNAs (hsa-let-7b, hsa-let-7g, hsa-miR-15b, hsa-miR-181b, hsa-miR-191, hsa-miR-200c, hsa-miR-26a, hsa-miR-27a, hsa-miR-30a-5p and hsa-miR-30c) were evaluated for their potential prognostic value in colorectal cancer patients. Forty eight snap frozen clinical colorectal samples (24 colorectal cancer and 24 paired normal patient samples) with detailed clinical follow-up information were selected. The expression levels of 10 miRNAs were quantified via qRT-PCR analysis. The statistical significance of these markers for disease prognosis was evaluated using a two tailed paired Wilcoxon test. A Kaplan-Meier survival curve was generated followed by performing a Logrank test. Among the ten miRNAs, hsa-miR-15b (p = 0.0278), hsa-miR-181b (p = 0.0002), hsa-miR-191 (p = 0.0264) and hsa-miR-200c (p = 0.0017) were significantly over-expressed in tumors compared to normal colorectal samples. Kaplan-Meier survival analysis indicated that hsa-miR-200c was significantly associated with patient survival (p = 0.0122). The patients (n = 15) with higher hsa-miR-200c expression had a shorter survival time (median survival = 26 months) compared to patients (n = 9) with lower expression (median survival = 38 months). Sequencing analysis revealed that hsa-miR-181b (p = 0.0098) and hsa-miR-200c (p = 0.0322) expression were strongly associated with the mutation status of the p53 tumor suppressor gene. Some of these miRNAs may function as oncogenes due to their over-expression in tumors. hsa-miR-200c may be a potential novel prognostic factor in colorectal cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2134920PMC
January 2006

Cellular cofactors affecting hepatitis C virus infection and replication.

Proc Natl Acad Sci U S A 2007 Jul 6;104(31):12884-9. Epub 2007 Jul 6.

Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, and Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY 10021, USA.

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.
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http://dx.doi.org/10.1073/pnas.0704894104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1937561PMC
July 2007

A mammalian microRNA expression atlas based on small RNA library sequencing.

Cell 2007 Jun;129(7):1401-14

Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Box 186, The Rockefeller University, New York, NY 10021, USA.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
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http://dx.doi.org/10.1016/j.cell.2007.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681231PMC
June 2007
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