Use of the Fluorescent Nucleoside Analogue Benzo[g]quinazoline 2′-O-Methyl--D-ribofuranoside to Monitor the Binding of the HIV-1 Tat Protein or of Antisense Oligonucleotides to the TAR RNA Stem-Loop

Abstract

Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday The Tat protein is an essential trans-activator of HIV gene expression. It interacts with its RNA recognition sequence, the trans-activation responsive region TAR, as well as cellular factors. These interactions are potential targets for drug discovery against HIV infection. We have developed a new and sensitive assay for the measurement of Tat binding to TAR in solution under equilibrium conditions based on the change of fluorescence of the base analogue benzo[g]quinazoline-2,4(1H,3H)-dione (BgQ) incorporated into the chemically synthesized model TAR stem-loop 2 to which was added Tat-[37-72] peptide (3). The results show that Tat-TAR binding strength is 2 ± 3-fold stronger than has previously been determined by mobility-shift analysis. Changes of fluorescence were used also to measure the binding of antisense 2'-O-methyloligonucleotides to TAR 2.1. Introduction. ± The human immunodeficiency virus type 1 (HIV-1) Tat protein is an essential trans-activator required for viral replication (for recent reviews, see [1 ± 3]). Tat interacts with an RNA sequence, the trans-activation responsive region TAR, a 59-residue stem-loop that occurs at the 5'-end of all viral RNA transcripts, as well as a Tat-associated kinase complex (TAK) that includes cyclin T1 and the kinase CDK9. The full mechanism of trans-activation by Tat is not yet established, but evidence from in vitro transcription techniques suggests that, in the absence of Tat, the transcription complex is unstable in the elongation phase leading to a predominance of short viral transcripts. When TAK becomes activated by Tat and TAR, the C-terminal domain of RNA polymerase II becomes hyperphosphorylated, an event that is essential for fulllength Tat-dependent transcription [4] [5]. The key virus-specific step in transactivation, however, is Tat/TAR recognition.Tat binds in vitro with high affinity near the apex of TAR to a region containing a 3-residue U-rich bulge (see below, Figs. 1 and 2). This interaction has been studied in great detail over many years (reviewed in [1]), including much work in our own laboratory on model TAR duplexes together with Tat protein made in E. coli or with synthetic Tat peptides [6] [7]. More recently, synthetic TAR duplexes have been used for cross-linking studies to Tat [8 ± 11]. Structural models of TAR either free or in the presence of a Tat peptide have also been obtained based on NMR characterization [12 ± 16], but no complete structure of the peptide/RNA complex has been produced.

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

“…Binding to the lower affinity site yielded an apparent microscopic K D of 300 ± 75 nM, and resulted in only a moderate decrease in fluorescence intensity. The observation of multiple binding events for Tat with ⌬TAR is consistent with previous studies (Weeks et al 1990;Weeks and Crothers 1992), and the observed K D 's fall within the range of those previously reported based on gel electrophoretic, filter, and fluorescencebinding assays performed under slightly different buffer, salt, and temperature conditions (Weeks et al 1990;Weeks and Crothers 1992;Churcher et al 1993;Arzumanov et al 2000). Titration of ⌬TAR-ap23 with the Tat peptide gave results that were quite similar to those described above, except that only about a fourfold initial increase in fluorescence intensity was observed before the latter began to decrease with increasing ligand concentration (data not shown).…”

“…To date, a few low molecular-weight compounds have been reported that block the Tat·TAR interaction in vitro with inhibition constants in the nano-to micromolar range (Mei et al 1995(Mei et al , 1997Hamy et al 1997Hamy et al , 1998Wang et al 1998;Sannes-Lowery et al 1999;Litovchick et al 2001;Du et al 2002;Lind et al 2002;Blount and Tor 2003;Hwang et al 2003;Davis et al 2004;Murchie et al 2004), some of which have also shown viral inhibition in cellular assays. Among these compounds are a number of amino acid and nucleotide analogs (Mestre et al 1999;Arzumanov et al 2000Arzumanov et al , 2001Mayhood et al 2000;Tamilarasu et al 2000) and aminoglycoside antibiotics (Mei et al 1995;Wang et al 1998;Blount and Tor 2003), which have been shown to effectively compete with Tat for binding to TAR. Studies have also focused on modifying and conjugating small molecules to enhance their specificity for TAR through directed and combinatorial chemical approaches (Hamy et al 1998;Gelus et al 1999;Litovchick et al 2001).…”

“…In addition, 2-AP fluorescence has been applied to characterize RNA conformational changes associated with peptide binding (Austin et al 2003;Barrick and Roberts 2003), chaperone mediate RNA refolding (Rist and Marino 2002), and ribozyme cleavage (Harris et al 2002;Jeong et al 2003). Fluorescence changes observed for another base analog, benzo[g]quinazoline-2,4(1H,3H)-dione incorporated at a position in the TAR bulge have also been used as a sensitive measure of Tat peptide binding (Arzumanov et al 2000). In general, the sensitivity of 2-AP's quantum yield to its microenvironment allows binding to be detected through direct contact, as well as indirectly through conformational changes in the RNA that may accompany the interaction with a ligand (Rist and Marino 2001).…”

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

“…This is the reverse situation compared with filter-binding experiments of Figure 2 Although the buffer conditions are not identical, it is of interest to note that the best 10-mer 1493 has a K d value (29 nM) similar to that previously measured by a direct equilibrium fluorescence method for a 12-mer OMe oligonucleotide binding to a stem-loop TAR RNA in a region with apical loop, double helix and internal bulge (approx. 20 nM) [34]. This suggests that the nucleotides bound within the A site may not be dissimilar in accessibility to those found within other structured RNAs and thus not significantly hindered by the overall 30 S ribosomal subunit nucleoprotein assembly.…”

Using an emissive uridine analogue for assembling fluorescent HIV-1 TAR constructs

“…24,25 Similarly, a TAR construct containing benzo[g]quinazoline-2,4(1H,3H)-dione, a large U analogue, displayed changes in fluorescence upon Tat binding when incorporated in the U-rich bulge. 26 We have recently reported a series of responsive fluorescent pyrimidine nucleoside analogues functionalized at the 5-position with aromatic five-membered heterocycles. 27 A furancontaining uridine is particularly useful.…”