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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Chem Biol Drug Des. 2011 Dec 22;79(2):202–208. doi: 10.1111/j.1747-0285.2011.01274.x

LIGAND-INDUCED CHANGES IN T BOX ANTITERMINATOR RNA STABILITY

S Zhou 1, G Acquaah-Harrison 1, KD Jack 1, SC Bergmeier 1, JV Hines 1,*
PMCID: PMC3466076  NIHMSID: NIHMS340847  PMID: 22117759

Abstract

The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (Tm) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner.

Keywords: antiterminator, Tm, riboswitch, tRNA, ligand screening

Introduction

Riboswitches are an intriguing and challenging target for drug discovery. These non-coding RNA regions are found in a wide variety of bacteria, including pathogenic bacteria, and respond to metabolic effector molecules to regulate transcription and translation of important bacterial genes (1, 2). The T box transcription antitermination riboswitch binds and structurally responds to non-aminoacylated (uncharged) cognate tRNA to regulate transcription of amino acid biosynthesis, transport and metabolism genes (3). The riboswitch is located within the 5′-untranslated region (5′-UTR) of the mRNA and forms specific base pairs with the tRNA both at the anticodon and at the acceptor end (Figure 1) (4, 5). The former base pairing confers the cognate specificity while the latter base pairing plays a direct role in the structural response of the riboswitch to the uncharged cognate tRNA (3).

Figure 1. T box riboswitch.

Figure 1

Schematic of T box riboswitch mechanism for a) transcription termination in the presence of excess aminoacylated cognate tRNA and b) transcription antitermination in the presence of excess non-aminoacylated cognate tRNA.

There are multiple conserved structural and sequence elements in the 5′-UTR of genes regulated by the T box riboswitch mechanism (68). It is the base pairing of the uncharged tRNA acceptor end nucleotides with four nucleotides in the bulge of an antiterminator element in the 5′-UTR, however, that turns the riboswitch “on” by stabilizing the antiterminator (9). The antiterminator element alone is thermodynamically less stable than an alternative structural element called the terminator. Consequently, without uncharged cognate tRNA present, the terminator forms preferentially and transcription is terminated shortly after the terminator is transcribed and before the protein coding region of the mRNA is reached (Figure 1b) (3). Since the antiterminator and terminator elements share common nucleotides, only one structural element can exist at a time. During transcription, the riboswitch binds uncharged cognate tRNA (if present in sufficient quantities) and the tRNA stabilizes the antiterminator before the full terminator sequences are transcribed (9, 10). In this manner, the riboswitch can respond to metabolic needs by switching “on” transcription of the entire mRNA in response to the metabolic signal of high levels of uncharged cognate tRNA.

The T box antiterminator RNA is an ideal target for drug discovery given the importance of the genes regulated by the T box riboswitch and the high sequence and secondary structure conservation of the antiterminator element (68). As a constitutively “off” riboswitch (i.e., one that is “off” unless the metabolic effector molecule binds), the T box riboswitch presents a unique challenge from a drug discovery perspective. The challenge is to develop ligands with high specificity and affinity that do not overly stabilize the antiterminator, or turn “on” the riboswitch, in a tRNA-independent manner. The goal is to find small molecules that disrupt tRNA binding without providing excessive stabilization to the antiterminator that might result in the ligand-antiterminator complex precluding the formation of the terminator element of the riboswitch. We have previously reported ligand classes that bind T box antiterminator RNA in a specific manner (1116). As part of a comprehensive drug discovery project focused on targeting the T box antiterminator RNA, we report in this paper 1,4-disubstituted 1,2,3-triazole ligands that affect antiterminator stability as determined using a novel, fluorescence-monitored thermal denaturation assay (Figure 2).

Figure 2. Fluorescence monitored thermal denaturation screening assay.

Figure 2

a) Schematic of fluorescence-monitored thermal denaturation assay. Upon thermal denaturation, the fluorophor switches from a quenched (open red circle) to unquenched (filled red circle) state. b) Antiterminator model RNA AM1A (labeled with Carboxyrhodamine (ROX) at the 5′-end and with Black-hole-quencher 2 (BHQ2) at the 3′-end). c) 1,4-disubstituted 1,2,3-triazole ligand

Methods and Materials

Synthesis and RNA Preparation

All 1,4-disubstituted 1,2,3-triazoles were prepared as previously described (14). Double-labeled T box antiterminator RNA 5′-Carboxyrhodamine-AM1A-3′-BlackHoleQuencher2 (5′ROX-AM1A-BHQ2, Figure 2b) was purchased from Trilink BioTechnologies, Inc. in a PAGE and RP-HPLC purified form and dialyzed in 10 mM NaH2PO4 pH 6.5, 0.01 mM EDTA prior to use.

Melting Temperature (Tm) assay development

A fluorescence-monitored thermal denaturation assay was developed to monitor Tm changes in a multiplex assay. To evaluate the relevance of the method, the Tm of 5′ROX-AM1A-BHQ2 was measured at different NaCl concentrations. The 5′ROX-AM1A-BHQ2 was renatured by heating to 90 °C for 1.5 min followed by slow cooling to room temperature. The Tm experiments consisted of 100 nM 5′ROX-AM1A-BHQ2 and various NaCl concentrations (0, 10, 20 and 50 mM) in 10 mM NaH2PO4 pH 6.5, 0.01 mM EDTA buffer. The samples were incubated in a Statagene Mx3000P qPCR instrument at 30 °C for 15 min at which point, the temperature was increased 1 degree and maintained for 3 minutes for each temperature step before recording the fluorescence (λex = 585 nm, λobs = 610 nm). A total of 61 temperature steps were completed to reach the final temperature of 90 °C. The Tm was determined from the first derivative of the data calculated using OD Deriv (17). For DMSO control experiments, the conditions were the same as described with the addition of 5% (v/v) DMSO in 10 mM NaH2PO4, 0.01 mM EDTA buffer. For comparison studies, UV-monitored thermal denaturation data was obtained as previously described (18).

Tm ligand screening

The fluorescence-monitored single ligand concentration Tm screening was measured using a Statagene Mx3000P qPCR instrument. Each experiment contained a final concentration of 100 nM 5′ROX-AM1A-BHQ2 and 100 μM ligand in 10 mM NaH2PO4, pH 6.5, 0.01 mM EDTA, 5% (v/v) DMSO buffer. Fluoresence-monitored thermal denaturation data of duplicate experiments were acquired as described above and the Tm was determined. The average AM1A melting temperature changes (ΔTm) were calculated using the equation ΔTm = Tm − Tm0, where the Tm0 is the AM1A melting temperature in the absence of ligand and Tm is the melting temperature in the presence of the ligand.

Results and Discussion

Multiplex thermal stability assay

Molecular beacons have been used in a variety of molecular biology applications, including real-time PCR, monitoring nucleic acid-protein interactions and in vivo RNA detection (19). The fluorescence-monitored thermal denaturation profile of molecular beacons has been used to monitor the stability and unfolding of nucleic acid structures (20) and to identify small molecules that could stabilize stem-loop RNA structures (21). A molecular beacon thermal denaturation profile is obtained by measuring the fluorescence intensity as a function of temperature. Due to the addition of fluorescent groups at the end of the nucleic acid stem region, the fluorescently measured Tm is often higher than the Tm measured by UV-monitored thermal denaturation. Different fluorophores and quenchers differentially contribute to the thermodynamic stability of the molecular beacons (22).

In this study, T box antiterminator model RNA AM1A was double-labeled with Carboxy-X-Rhodamine (ROX) at the 5′ end and Black Hole Quencher 2 (BHQ2) at the 3′ end to construct molecular beacon 5′ROX-AM1A-BHQ2 (Figure 2b). ROX was selected because its quantum yield does not change significantly with increasing temperature (23). In order to achieve the maximum quenching efficiency, the fluorophor-quencher pair should have adequate spectral overlap. Thus, BHQ2 was selected due to the good spectral overlap between the BHQ2 absorbance spectrum and the ROX emission spectrum (23).

The fluorescence-monitored Tm assay was based on the premise that when the AM1A exists in its native secondary structure, the A1 helix conformation brings ROX and BHQ2 in close proximity where the fluorescence of ROX is effectively quenched. As the temperature increases, the nucleic acid starts to denature from the native secondary structure to a random linear polynucleotide (24). During this process ROX and BHQ2 are separated from each other, leading to the observed increase in fluorescence (Figure 2a).

The validity of monitoring ligand-induced stabilization using the fluorescently labeled AM1A was evaluated by comparison to unlabeled AM1A and by monitoring the effect of conditions known to affect RNA stability. Results are shown in Figure 3 and summarized in Table 1. The fluorescence-monitored Tm observed for ROX-AM1A-BHQ2 (71°C) was higher than that observed by UV-monitored thermal denaturation (24) of unlabeled AM1A in the same buffer (Tm = 51°C, Supplementary Material). This is likely due to the end labeled fluorophor and quencher pair stacking on the first base pair of the double helix. This effect has been observed in similar nucleic acid fluorophor/quencher constructs and contributes to the overall structural stability of the nucleic acid (25).

Figure 3. Tm model study of fluorescently labeled antiterminator model RNA AM1A.

Figure 3

Fluoresence-monitored thermal denaturation of ROX-AM1A-BHQ2 in the presence of a) differing concentrations of Na+ and b) 5% (v/v) DMSO where dF/dT is the change in fluorescence intensity at 610 nm as a function of change in temperature. The Tm is the temperature at maximal dF/dT.

Table 1.

Tm (°C) of ROX-AM1A-BHQ2 determined using fluorescence-monitored thermal denaturation method.a

[NaCl] (mM) DMSO (v/v) Tm (°C)
0 - 71.3
10 - 73.5
20 - 75.5
50 - 80.4
0 5% 71.2
a

Tm measured in 10 mM NaHPO4, pH 6.5, 0.01 mM EDTA with 100 nM ROX-AM1A-BHQ2

Monovalent cations are known to stabilize RNA secondary structures and increase the observed Tm (26). As expected, an increase in the concentration of Na+ resulted in an increase in the Tm observed by the fluorescence-monitored thermal denaturation method (Figure 3a and Table 1). The Na+-dependent shift in Tm for unlabeled AM1A is comparable (Supplementary Material), indicating that conjugation with the fluorophore and quencher does not affect the dependence of AM1A stability on buffer ionic conditions. Overall, the data indicate that the stability of the ROX-AM1A-BHQ2 is suitably responsive to environmental conditions and that the fluorescence monitored ΔTm can be utilized in screening for ligand-induced stabilization.

Since DMSO is known to denature nucleic acids at high concentrations (27), the effect of 5% (v/v) DMSO on the Tm was tested to determine if the ligand stock solution solvent would affect the Tm results. No significant change in the Tm was observed when DMSO was present (Figure 3b and Table 1) indicating that the relatively low final concentration of DMSO in the assay buffer does not interfere with the results.

Previous studies have indicated that Mg2+ facilitates both complete tRNA binding to antiterminator model RNA (28) and in vitro transcription antitermination (4). Consequently, we wished to investigate the effect of Mg2+ on the fluorescently monitored thermal denaturation. However, at 5 mM Mg2+, the minimal concentration used in tRNA-antiterminator binding studies (29), the ROX-AM1A-BHQ2 was so stabilized that a Tm could not be determined due to insufficient denaturation even at the highest temperature (Supplementary Material). The fluorophore/quencher labels stabilize the antiterminator model RNA to such an extent that it is a challenge to determine the Tm even with just the addition of monovalent cations (e.g., 50 mM NaCl, figure 3a). While this precludes the use of the fluorescently monitored Tm assay in studying the tRNA-antiterminator complex, the low salt conditions of the assay are still relevant for looking at small molecule-RNA interactions. The structure of the antiterminator model RNA is not significantly dependent on Mg2+ nor salt until ≥ 100 mM NaCl and even then, the excess salt only results in selective stabilization of one of multiple conformers that is already present at lower salt concentrations (30).

Ligand binding site implications

The ligand-induced ΔTm of 1,4-disubstituted 1,2,3-triazoles was determined at 100 μM, the same concentration previously used to screen for antiterminator binding (14). The majority of the compounds either had no effect on the Tm at the concentration tested or led to a slight increase in the Tm (Table 2). The compounds that most stabilized ROX-AM1A-BHQ2 (positive ΔTm) were GHB-7, GHB-74, GHB-75, GHB-77, GHB-109. The compound that most destabilized the antiterminator was GHB-110. This is consistent with other ligand-induced ΔTm RNA studies. Typically, if there is an effect, ligands that bind RNA tend to stabilize the RNA secondary structure, as determined by UV-monitored thermal denaturation. There are instances, however, where ligands have been shown to destabilize the secondary structure, resulting in a reduction in the observed Tm (31). Interestingly, there is evidence for very structure-specific ligand-induced Tm effects since the ΔTm for GHB-7 was 3.2°C while GHB-26 had no significant effect on the Tm (ΔTm=0.2°C). These two compounds differ only by a methylene group within NR2 suggesting that the exact length of the alkyl phenyl chain (and thereby placement of the functional group when bound to AM1A) may play a role in ligand-specific binding and stabilization of the RNA.

Table 2.

Ligand-induced ΔTm (°C) of ROX-AM1A-BHQ2a

Ligand NR2 R1 R2 R3 ΔTmb
GHB-7 N(CH3)(CH2)3Ph H CH2OC(O)NHCH2Ph H 3.2
GHB-9 N(CH3)(CH2)3Ph H n-C4H9 H 0.7
GHB-10 4-Ph-piperazine H (CH2)4 −0.5
GHB-11 3-(CO2Et)-piperidine H n-C4H9 H −0.2
GHB-12 N(CH3)CH2(c-C6H11) H n-C4H9 H −0.2
GHB-13 4-Ph-piperazine H n-C4H9 H −0.2
GHB-14 3-(CO2Et)-piperidine H (CH2)4 −0.3
GHB-15 N(CH3)(CH2)2CH(CH3)2 H (CH2)4 −0.7
GHB-16 N(CH3)(CH2)2CH(CH3)2 H n-C4H9 H −0.2c
GHB-17 N(CH3)(CH2)2Ph H (CH2)4 −0.3
GHB-18 N(n-C4H9)2 H (CH2)4 0.4c
GHB-19 4-Ph-piperidine H n-C4H9 H −0.2
GHB-20 4-Ph-piperidine H (CH2)4 0.3
GHB-21 N(CH3)CH2(c-C6H11) H (CH2)4 −0.8
GHB-22 4-Ph-piperazine H Ph H 0.9
GHB-23 N(CH3)(CH2)2CH(CH3)2 H Ph H −0.7
GHB-24 4-Ph-piperazine H CH2OCH2Ph H 0.4
GHB-25 3-(CO2Et)-piperidine H CH2OCH2Ph H 0.6
GHB-26 N(CH3)(CH2)2Ph H CH2OC(O)NHCH2Ph H −0.2
GHB-27 N(n-C4H9)2 H CH2OC(O)NHCH2Ph H −0.2
GHB-28 3-(CO2Et)-piperidine H CH2OC(O)NHCH2Ph H −0.2
GHB-29 N(CH3)(CH2)2CH(CH3)2 H CH2OC(O)NHCH2Ph H 0.7
GHB-30 N(CH3)CH2(c-C6H11) H CH2OC(O)NHCH2Ph H −0.3
GHB-31 4-Ph-piperidine C(O)Ph (CH2)4 −0.3
GHB-32 N(CH3)CH2(c-C6H11) C(O)Ph (CH2)4 −0.2
GHB-33 N(CH3)(CH2)3Ph C(O)CH2OPh (CH2)4 −0.2
GHB-34 4-Ph-piperidine C(O)CH2OPh (CH2)4 0.3
GHB-35 4-Ph-piperazine C(O)CH2OPh (CH2)4 −0.2
GHB-36 N(CH3)(CH2)2Ph C(O)CH2OPh (CH2)4 0.4
GHB-37 N(n-C4H9)2 C(O)CH2OPh (CH2)4 0.4
GHB-38 3-(CO2Et)-piperidine C(O)CH2OPh (CH2)4 0.8
GHB-39 N(CH3)CH2(c-C6H11) C(O)CH2OPh (CH2)4 0.7
GHB-40 4-Ph-piperazine H CH2OC(O)NH(n-C4H9) H −0.3
GHB-41 4-Ph-piperazine H CH2OC(O)Ph H −0.3
GHB-42 morpholine H CH2OC(O)NH(n-C4H9) H −0.2
GHB-43 morpholine H CH2OC(O)Ph H −0.1
GHB-44 N(n-C4H9)2 C(O)CH2Ph (CH2)4 0.3
GHB-45 N(CH3)CH2(c-C6H11) C(O)CH2Ph (CH2)4 0.4
GHB-46 morpholine H CH2OC(O)c-C6H11 H 0.4
GHB-47 morpholine C(O)CH2OPh (CH2)4 0.9
GHB-48 3-(CO2Et)-piperidine C(O)CH2Ph (CH2)4 0.7
GHB-49 3-(CO2Et)-piperidine C(O)Ph (CH2)4 −0.3
GHB-50 N(CH3)CH2Ph H Ph H −0.1
GHB-51 N(CH3)(CH2)2CH(CH3)2 C(O)Ph (CH2)4 0.9
GHB-57 4-Ph-piperazine H CH2OC(O)NHCH2Ph H 0.7
GHB-58 4-Ph-piperazine C(O)Ph (CH2)4 −0.3
GHB-59 4-Ph-piperazine H CH2OC(O)n-C7H15 H −0.3
GHB-60 N(CH3)(CH2)2CH(CH3)2 C(O)CH2OPh (CH2)4 −0.3
GHB-61 4-Ph-piperidine H CH2OC(O)NHCH2Ph H 0.2
GHB-62 morpholine H (CH2)4 0.3
GHB-63 morpholine H n-C4H9 H −0.1
GHB-64 morpholine H Ph H −0.1
GHB-65 N(CH3)CH2(c-C6H11) H CH2OCH2Ph H 0.4
GHB-66 morpholine H CH2OCH2Ph H 0.8
GHB-67 N(n-C4H9)2 H CH2OC(O)NH(n-C4H9) H 0.7
GHB-68 3-(CO2Et)-piperidine H CH2OC(O)NH(n-C4H9) H −0.3
GHB-69 N(CH3)(CH2)2CH(CH3)2 H CH2OC(O)NH(n-C4H9) H −0.3
GHB-70 N(CH3)CH2(c-C6H11) H CH2OC(O)NH(n-C4H9) H −0.3
GHB-71 N(n-C4H9)2 H CH2OC(O)c-C6H11 H 0.7
GHB-72 3-(CO2Et)-piperidine H CH2OC(O)c-C6H11 H 0.7
GHB-73 N(CH3)(CH2)2CH(CH3)2 H CH2OC(O)c-C6H11 H 0.7
GHB-74 N(CH3)CH2(c-C6H11) H CH2OC(O)c-C6H11 H 1.6
GHB-75 N(CH3)(CH2)3Ph H CH2OC(O)n-C7H15 H 4.1c
GHB-76 N(n-C4H9)2 H CH2OC(O)n-C7H15 H 0.3
GHB-77 N(CH3)(CH2)3Ph H CH2OC(O)Ph H 1.6
GHB-78 N(CH3)(CH2)2Ph H CH2OC(O)Ph H 0.7
GHB-79 N(n-C4H9)2 H CH2OC(O)Ph H −0.2
GHB-80 3-(CO2Et)-piperidine H CH2OC(O)Ph H 0.7
GHB-81 N(CH3)(CH2)2CH(CH3)2 H CH2OC(O)Ph H 0.7
GHB-82 N(CH3)CH2(c-C6H11) H CH2OC(O)Ph H 0.7
GHB-83 morpholine C(O)Ph (CH2)4 0.2
GHB-84 morpholine C(O)CH2Ph (CH2)4 −0.4
GHB-107 morpholine H CH2OC(O)n-C7H15 H 0.1
GHB-108 N(CH3)(CH2)2Ph H n-C4H9 H −0.2d
GHB-109 N(n-C4H9)2 H n-C4H9 H 1.1
GHB-110 3-(CO2Et)-piperidine H Ph H −0.9
GHB-111 N(CH3)CH2(c-C6H11) H Ph H 0.1
GHB-112 N(CH3)(CH2)3Ph H CH2OCH2Ph H 0.1
GHB-113 N(CH3)(CH2)2Ph H CH2OCH2Ph H 0.1
a

All compounds (100 μM) were assayed in 10 mM NaH2PO4, pH 6.5, 0.01 mM EDTA, 5% (v/v) DMSO with 100 nM ROX-AM1A-BHQ2. λex = 585 nm, λem = 610 nm, heating rate = 0.33 °C/min.

b

Average ΔTm values where ΔTm = Tm − Tm0 and standard deviation of duplicate experiments ≤ 0.5°C unless otherwise noted.

c

Standard deviation of duplicate experiments ≤ 0.6°C

d

Standard deviation of duplicate experiments ≤ 0.8°C

Conclusion & Future Directions

The results from this study indicate that the fluorescence-monitored thermal denaturation assay is a useful method for screening ligands to determine whether or not they stabilize the T box antiterminator RNA element. More importantly, through the use of this assay, several 1,4-disubstituted 1,2,3-triazoles were identified that significantly altered the observed stability of the antiterminator model RNA AM1A in a structure-dependent manner. A ligand-induced ΔTm does not, a priori, indicate that the ligand will be effective at disrupting the tRNA-antiterminator RNA complex, however, it is very intriguing that GHB-7, one of the compounds with the greatest shift in Tm, is also one of the compounds that resulted in the greatest disruption of the tRNA-antiterminator complex in a fluorescence anisotropy assay (32). Further studies are in progress to characterize the interactions of the lead triazole compounds with AM1A in order to determine what specific structural features lead to stabilization or destabilization of the antiterminator model RNA. This information will be vital for the development of potential antibacterial agents that target the T box riboswitch.

Supplementary Material

Supp Fig S1-S3

Acknowledgments

We thank the NIH (GM073188, GM61048) and Ohio University, through the BioMolecular Innovation & Technology (BMIT) project, for support of this work.

Footnotes

Supplmentary Material

UV-monitored thermal denaturation data is provided in the Supplementary Material.

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