Spotted Long Oligonucleotide Arrays for Human Gene Expression Analysis

DNA microarrays produced by deposition (or `spotting')of a single long oligonucleotide probe for each gene may be an attractive alternative to other types of arrays. We produced spotted oligonucleotide arrays using two large collections of ∼70-mer probes, and used these arrays to analyze gene expression in two dissimilar human RNA samples. These samples were also analyzed using arrays produced by in situ synthesis of sets of multiple short (25-mer)oligonucleotides for each gene (Affymetrix GeneChips). We compared expression measurements for 7344 genes that were represented in both long oligonucleotide probe collections and the in situ-synthesized 25-mer arrays. We found strong correlations (r = 0.8–0.9)between relative gene expression measurements made with spotted long oligonucleotide probes and in situ-synthesized 25-mer probe sets. Spotted long oligonucleotide arrays were suitable for use with both unamplified cDNA and amplified RNA targets, and are a cost-effective alternative for many functional genomics applications. Most previously reported evaluations of microarray technologies have focused on expression measurements made on a relatively small number of genes. The approach described here involves far more gene expression measurements and provides a useful method for comparing existing and emerging techniques for genome-scale expression analysis

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs

<div><p>Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity <i>K</i>_<i>app</i> was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.</p></div

Inhibition of influenza virus and crude replication complex.

<p>Inhibition of influenza virus and crude replication complex.</p

Determination of apparent binding affinity (<i>K</i>_app) of GTP, T-1106-TP, and 4’vinyl-FdGTP during single nucleotide incorporation by the recombinant influenza RdRp.

<p>(A) Diagram illustrating formation of RNA 9-mer product from ApG primer and further elongation to RNA 14-mer product; (B) Summary of <i>K</i>_app and selectivity for GTP and analogs; Incorporation of GTP (panel C), T-1106-TP (panel D), and 4’vinyl-FdGTP (panel E) as a function of NTP analog concentration. The product formation is plotted as a function of compound concentration for GTP (panel F), T-1106-TP (panel G), and 4’vinyl-FdGTP (panel H), respectively. The calculated <i>K</i>_<i>app</i> values for GTP, T-1106-TP, and 4’vinyl-FdGTP are 9 nM, 27 nM, and 29 μM respectively.</p

Mechanism of action for different nucleotide analogs during recombinant RdRp-catalyzed RNA synthesis.

<p>(A) Reaction scheme showing cleavage of the ³³P-m⁷G¹67 primer by influenza polymerase heterotrimer, base pairing with miniHA template, and expected transcription product. (B) Reaction mixtures contained miniHA template, radiolabeled capped RNA primer ³³P-m⁷G¹67, 500 μM GTP or GTP analogs, and 500 μM for each of natural CTP, UTP, and ATP. Products were separated by 25% PAGE and visualized by autoradiography. The top of the gel was illustrated to show the formation of long transcription products. The ladder of nucleotide bases on the side bar demonstrates the correct incoming nucleotide base-pairing with the miniHA template.</p

Activity of RNA replication and transcription by recombinant influenza RdRp heterotrimer.

<p>(A) The rate of ³³P-α-GTP incorporation by cRNP is proportional to the estimated viral particles used to generate cRNP; (B) The rate of ³³P-α-GTP incorporation by recombinant RdRp is proportional to the PA/PB1/PB2 trimer protein concentration; (C and D) The product formation presented by an arbitrary unit on the phosphor imager is linear with time and similar between the replication and transcription rates observed with cRNP (panel C) and recombinant RdRp (panel D) during ApG-primed RNA replication (●) or rabbit globin mRNA-primed RNA transcription (■). (E) Product formation using different RNA templates was analyzed on a 10% polyacrylamide gel. In contract to cRNP-catalyzed RNA synthesis (far left lane) which generated a smeared ladder of long products at the top of the gel, recombinant RdRp generated approximately template length products during ApG-primed RNA replication (D, B). Furthermore, transcripts of PA-30 and mini-HA containing an additional 9–11 nucleotides were generated during rabbit globin mRNA-primed RNA transcription (C, A).</p

Characterization of a KDM5 small molecule inhibitor with antiviral activity against hepatitis B virus

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases