1048–1056 Nucleic Acids Research, 2010, Vol. 38, No. 3 Published online 20 November 2009 doi:10.1093/nar/gkp1022 A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity Alexander S. Wahba1, Abbasali Esmaeili2, Masad J. Damha1 and Robert H. E. Hudson3,* 1Department of Chemistry, McGill University, Montreal, QC, H3A 2K6 Canada, 2Department of Chemistry, University of Birjand, Birjand, Iran and 3Department of Chemistry, University of Western Ontario, London, ON, N6A 5B7 Canada Downloaded from https://academic.oup.com/nar/article/38/3/1048/3112336 by guest on 27 September 2021 Received August 15, 2009; Revised and Accepted October 19, 2009 ABSTRACT identified as a potential target for antiretroviral therapy as it is required for virus infectivity (3); yet there are no 6-Phenylpyrrolocytidine (PhpC), a structurally con- antiRNase H agents in clinical use. Few inhibitors of HIV- servative and highly fluorescent cytidine analog, 1 RNase H were identified until the transition of testing was incorporated into oligoribonucleotides. The methods from gel-based techniques to fluorescent assays PhpC-containing RNA formed native-like duplex amenable to high-throughput screening (HTS) (4–8). The structures with complementary DNA or RNA. The most widely used assay was developed by Parniak and co- PhpC-modification was found to act as a sensitive workers (6) and utilizes a two label, molecular beacon reporter group being non-disruptive to structure and strategy (9) in which the RNA strand is labeled with a 0 the enzymatic activity of RNase H. A RNA/DNA 3 -terminal fluorophore (fluorescein, F) and a DNA 0 hybrid possessing a single PhpC insert was an strand with a quencher (dabcyl, Q) at the 5 -terminus excellent substrate for HIV-1 RT Ribonuclease H (Scheme 1). Fluorescent studies involving nucleic acids most often and rapidly reported cleavage of the RNA strand utilize luminescent tags such as fluorescein or rhodamine with a 14-fold increase in fluorescence intensity. sometimes in combination with a quencher such as The PhpC-based assay for RNase H was superior 4-(dimethylaminoazo)benzene (dabcyl). However, these to the traditional molecular beacon approach in probes can be perturbing to the processes under investiga- terms of responsiveness, rapidity and ease (single tion due to the steric bulk or non-polar groups they intro- label versus dual). Furthermore, the PhpC-based duce to DNA and RNA. Through our own studies on assay is amenable to high-throughput microplate inhibitors of HIV-1 RNase H, we have observed that the assay format and may form the basis for a new 50-dabcyl quencher substantially reduces the catalytic effi- screen for inhibitors of HIV-RT RNase H. ciency of RNase H for its RNA/DNA substrate (SI). The use of intrinsically fluorescent nucleotide base analogs offers a more conservative approach to fluores- INTRODUCTION cence labeling of nucleic acids. Using phosphoramidite The continuing emergence of drug resistant variants of the solid-phase synthesis (10), a fluorescent nucleotide can human immunodeficiency virus (HIV) calls for new tools be incorporated at any position on an oligomer without to combat this epidemic (1). Current clinical treatment of the use of linkers or postsynthetic modifications. In HIV targets essential enzymatic activities associated with addition, the fluorescence intensity of many base the reverse transcription of the viral genome and the modified nucleotides are responsive to changes in their protease activity required for maturation of protein microenvironment, making them reporters for nucleic components of the virus. The most effective control of acid structure and dynamics (11). Despite these advan- the disease has been achieved with combination therapy tages, few fluorescent nucleobase analogs have found targeting these pathways simultaneously. The inclusion of widespread use as a tool for molecular biology, additional targets in a combination drug regimen is highly 2-aminopurine (12) and the pteridine (13) base analogs desirable for potent and long-term disease management. being historically the most important. Recent work from The ribonuclease H (RNase H) activity associated with Tor and others have elegantly demonstrated the value and HIV-reverse transcriptase (HIV-RT) degrades the viral utility in the design and discovery of new fluorescent RNA genome in RNA/DNA hybrids (2), and has been nucleobases (14–17). Despite these and other recent *To whom correspondence should be addressed. Tel: +519 661 2111 (Ext. 86349); Fax: +519 661 3022; Email: [email protected] ß The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2010, Vol. 38, No. 3 1049 Table 1. Oligonucleotides used in RNase H assays and Tm data 0 0 a b Name Sequence (5 –3 ) Tm ( C) DNA-1 RNA-2 RNA-1 GAU CUG AGC CUG GGA GCU 65.1 78.5 PhpC-1 GAU CUG AGC CUG GGA G*CU 64.7 78.8 PhpC-2 GAU CUG AGC *CUG GGA GCU 67.5 80.8 aPhpC indicated by *C. b Tm values represent the average of at least 3 independent experiments within 1C. Scheme 1. Representation of fluorescent RNase H assay using a dual label system employing a fluorophore and a quencher. Downloaded from https://academic.oup.com/nar/article/38/3/1048/3112336 by guest on 27 September 2021 Figure 1. The structure of cytidine (C) and unsubstituted pC, with Scheme 2. Reporting via a single fluorescent nucleotide analogs. numbering, compared to PhpC. R = ribose or 20-deoxyribose. *C = 6-phenylpyrrolocytidine (PhpC). advances, there remains a paucity of intrinsically fluores- Table 1), we replaced a single rC nucleoside with PhpC cent cytidine analogs that demonstrate responsiveness to close to the 30-terminus of the RNA/DNA hybrid, their microenvironment and state of hybridization (18) PhpC-1, where it exhibits low fluorescence (quenched thus motivating this work. state) as depicted in Scheme 2. Treatment with HIV-1 The success of a fluorescent nucleobase analog as a RT RNase H generates an RNA tetranucleotide (25) useful reporter resides in its ability to form proper bearing PhpC, and quickly dissociates from its DNA Watson–Crick base pairs, thermostability in double complement with a concomitant dramatic increase in stranded nucleic acids, changes in fluorescence with differ- fluorescence emission. ent hybridization states, recognition of nucleic acid processing/binding enzymes, and its fluorescence intensity. The cytidine analogs pyrrolocytidine (pC, Figure 1) with MATERIALS AND METHODS 6-methyl substitution (MepC) (19–21) satisfies the above 0 0 criteria as a fluorescent reporter, though its fluorescence 5 -O-(4,4 -dimethoxytrityl)-5-iodouridine (2) intensity, as manifested by a modest quantum yield (È), Uridine (2.44 g, 10 mmol) was added to a mixture of lags behind competing chemistries ultimately making iodine (2.54 g, 10 mmol) and silver sulfate (3.12 mg, it a less sensitive probe. One of our laboratories has 10 mmol) in methanol (200 ml) at room temperature shown that the low quantum yield of MepC can be (r.t.). The mixture was stirred for 8 min, after which time remedied by substituting the C-6 position with an the yellow solid was removed by filtration and the filtrate aromatic group without any penalties on sensitivity or was evaporated under reduced pressure. The residue was base pairing fidelity (22–24). co-evaporated with 2 : 1 MeOH/H2O (50 ml) to remove We have now synthesized the ribonucleoside of iodine (which sublimed away) and crystallized from 6-phenylpyrrolocytidine (PhpC, Figure 1) and found water to give pure 5-iodouridine as a white solid (4.44 g, that, like its DNA and PNA homologs, it ranks among 60%). 5-Iodouridine (2.22 g, 6 mmol) was dissolved in the brightest C-analog luminiophores. The fluorescence of pyridine (30 ml) and 4,40-dimethoxytritylchloride (2.64 g, PhpC is sensitive to its microenvironment, and displays 7.8 mmol) was added in four portions during a 4 h a max excitation (360 nm) and max emission (466 nm) period. After an additional 2 h of stirring at r.t. the significantly red-shifted from the absorption of other reaction was quenched by adding 1 ml of 5% NaHCO3 biomolecules. The combination of these properties for 15 min. The solution was concentrated under reduced makes PhpC unique among fluorescent nucleobase pressure, redissolved in 50 ml of dichloromethane (DCM) analogs and has enabled its use as a fluorescent reporter and washed with a solution of 5% NaHCO3 (30 ml). The group in an RNase H assay. Maintaining the same RNA product was further extracted with two 25 ml portions of sequence as the traditional FQ-based assay (6) (RNA-1, DCM, and the combined organic layer was dried over 1050 Nucleic Acids Research, 2010, Vol. 38, No. 3 0 0 0 Mg2SO4 and evaporated to leave the crude product. The (C-4 ), 75.67 (C-2 ), 68.72 (C-3 ), 61.14 (ÀCH2O), 60,29 0 residue was then purified by silica gel column chromatog- (C-5 ), 55.45 (2CH3OAr). ESI-TOF (m/z) 669.2 (M + Na). raphy using an elution gradient with DCM/MeOH/TEA (98 : 1 : 1 to 96 : 3 : 1). Fractions containing 2 were evaporated to give a white foam (3.72 g, 55%). 1H NMR 50-O-(4,40-dimethoxytrityl)-6-PhpC (5) (400 MHz, CDCl3): d = 8.14 (s, 1 H), 7.21–7.42 (m, 11 H), A pressure bottle with compound 4 (0.64 g, 1.0 mmol), 6.84 (m, 4 H), 6.31 (dd, 1J = 6.8 Hz, 2J = 5.9 Hz, 1 H), concentrated NH3OH (4 ml) and MeOH (6 ml) was 4.55 (m, 1 H), 4.07 (m, 1 H), 3.79 (s, 6 H), 3.78 (m, 1 H), stirred at 55C for 24 h.