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Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat; General factors, general transcription accessory proteins; Antibody, anti-RNA:DNA; PK, proteinase K; Oligos, DNA o-nts complementary to target RNA.

Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat; General factors, general transcription accessory proteins; Antibody, anti-RNA:DNA; PK, proteinase K; Oligos, DNA o-nts complementary to target RNA.

Drug Discovery Diagram

Figure 9 The figure shows data for three different transcription reactions: (1) basal (right inclined stripes), (2) Tat-activated (solid), and (3) a-amanitin treated (left-inclined stripes). (A) Dependence of the in vitro transcription signal on the addition of cDNA o-nts. (B) Dependence of the Tat-activated signal on RNase T1. First set of columns, RNase T1 was added to the reaction mixture after transcription took place; second set of columns, RNase T1 was added to the reaction mixture prior to the addition of nuclear extract and Tat; third set of columns, RNase T1 was omitted from the reaction. Error bars represent the standard deviation of three experiments.

Figure 9 The figure shows data for three different transcription reactions: (1) basal (right inclined stripes), (2) Tat-activated (solid), and (3) a-amanitin treated (left-inclined stripes). (A) Dependence of the in vitro transcription signal on the addition of cDNA o-nts. (B) Dependence of the Tat-activated signal on RNase T1. First set of columns, RNase T1 was added to the reaction mixture after transcription took place; second set of columns, RNase T1 was added to the reaction mixture prior to the addition of nuclear extract and Tat; third set of columns, RNase T1 was omitted from the reaction. Error bars represent the standard deviation of three experiments.

G-less cDNA o-nts were not added to the reaction mixture, the signals obtained for both basal and Tat-activated transcription were identical to background levels (that is, the a-amanitin treated sample).

Tat activates the transcription of HIV-1 genes by binding to the TAR sequence present at the 5' end of nascent HIV-1 transcripts. Therefore degradation of TAR-RNA by RNAse T1 treatment should abolish transcriptional activation by Tat. RNase T1 degrades the RNA at guanosine residues. Figure 9B shows that the Tat signal is dependent on the presence of the TAR element. Addition of RNase T1 to the in vitro transcription reaction after transcription took place yielded the maximal Tat-induced signal, and addition of RNase T1 before the transcription reaction reduced the signal to basal transcription level. In the absence of RNase T1, the overall basal and Tat-activated signals were reduced. This signal reduction presumably results from the formation of secondary structure in the RNA transcript, which interferes with hybridization to the cDNA o-nts. Thus, in order to achieve the maximal signal, all assays were performed with RNase T1 treatment following the transcription reaction. Further confirmation of the dependence of the Tat signal on the TAR element was obtained by measuring the signal generated with a mutant version of the LTR promotor from which the TAR element was removed. Transcription from this mutated promotor yielded a signal equivalent to basal levels.

Tat induced transcription to maximal levels at about 100 nM, and this signal was completely abolished by a-amanitin in the reaction (Fig. 10A). The Tat concentration chosen (40 nM) for the HTS assays was in the linear region of the curve. The nucleoside analog DRB (panel B), which is a well-characterized inhibitor of Tat activation [45], inhibits the Tat signal with an IC50 of 1 |M (Fig. 10B). A similar IC50 has been obtained with a gel-based transcription assay. Maximal Tat-activated and basal signals were obtained with 150 |M of each of the four NTPs (Fig. 10C). The signal obtained in the absence of NTPs was identical to the background levels observed with a-amanitin-treated samples. All HTS assays were performed with 250 | M of each NT. The signal reached saturation when the DNA template (LTR#5) amounts greater than 0.25 |g were added to the in vitro transcription reaction (Fig. 10D). For the HTS assays, 0.75 to 1 |g of the DNA template was used.

A. Robotic Assay of RNAPII In Vitro Transcription

After optimization of the assay conditions, a HTS robotic assay designed to identify specific inhibitors of Tat function was performed. The robotic assay was performed on a Zymark robot. The assay consists of seven reagent addition steps and one wash step. The additions were performed by the Zymark pipettor arm, the Zymark RAS-RAM unit, or a Titertek multidrop from ICN (Costa Mesa, CA). The wash was performed with a 96-well microtiter plate washer from Bio-Tek Instruments (Winooski, VT). During all incubation steps, the microtiter reaction plate was placed on a shaker. About fifty 96-well plates could be assayed in less than 10 hr.

Figure 11 shows representative data obtained from a single robotic run. The signals observed in the first eight wells of the microtiter plate corresponded

Figure 10 Effect of various assay parameters on Tat activation of HIV-1 in vitro transcription. (A) Dose-response of Tat with and without a-amanitin. (B) Inhibition by DRB of basal and Tat-activated (40 nM) transcription signal. (C) Dose-response of NTP mix (mix of ATP, CTP, GTP, and UTP) on basal and Tat-activated (20 nM) transcription. (D) Dose-response of the DNA template (LTR#5) on basal and Tat-activated (20 nM) transcription.

Figure 10 Effect of various assay parameters on Tat activation of HIV-1 in vitro transcription. (A) Dose-response of Tat with and without a-amanitin. (B) Inhibition by DRB of basal and Tat-activated (40 nM) transcription signal. (C) Dose-response of NTP mix (mix of ATP, CTP, GTP, and UTP) on basal and Tat-activated (20 nM) transcription. (D) Dose-response of the DNA template (LTR#5) on basal and Tat-activated (20 nM) transcription.

to basal transcription reaction. The signal observed in the last eight wells (wells 89 to 96) corresponded to a-amanitin-treated samples. The other 80 wells received all of the reagents necessary for Tat-activated transcription, along with a different test drug compound in each well at a concentration of 10 |M (dissolved in DMSO). The DMSO concentration in all the wells was 5%. Typically, a five-to tenfold window was observed between the wells with (activated) and without (basal) Tat. The standard deviation from the mean was generally less than 20% for assays containing pure chemicals. These features make the assay suitable for automated HTS. Over 200,000 chemicals were screened using this technology. The assay format described herein is a general one and is completely independent of the transcriptional apparatus. Therefore it can in principle be used to develop a HTS assay for any transcriptional system.

Figure 11 Robotic data obtained from in vitro transcription reactions performed in a 96-well microtiter plate containing 80 random pure chemicals. Columns 1 to 8 correspond to basal transcription reactions; columns 89 to 96 correspond to a-amanitin-treated samples. The other 80 columns correspond to wells that received all of the reagents necessary for Tat-activated transcription along with a pure chemical at a concentration of 10 ||M. The DMSO concentration in all the wells was 5%.

Figure 11 Robotic data obtained from in vitro transcription reactions performed in a 96-well microtiter plate containing 80 random pure chemicals. Columns 1 to 8 correspond to basal transcription reactions; columns 89 to 96 correspond to a-amanitin-treated samples. The other 80 columns correspond to wells that received all of the reagents necessary for Tat-activated transcription along with a pure chemical at a concentration of 10 ||M. The DMSO concentration in all the wells was 5%.

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