A reverse transcriptase stop assay revealed diverse quadruplex formations in UTRs in mRNA Masaki Hagihara a , Keisuke Yoneda a , Hiroaki Yabuuchi b , Yasushi Okuno b , Kazuhiko Nakatani a, * a Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research (ISIR), Osaka University, Ibaraki 567-0047, Japan b Department of Systems Bioscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan article info Article history: Received 30 November 2009 Revised 21 January 2010 Accepted 25 January 2010 Available online 17 February 2010 Keywords: RNA quadruplex Reverse transcriptase mRNA abstract Here, we developed a reverse transcriptase based method (RTase stop assay) to characterize quadruplex formations in guanine-rich RNAs with high sensitivity and specificity. By using the RTase stop assay, we also revealed a plausible structural polymorphism in biologically important RNAs. The RTase stop assay would provide helpful insight into RNA quadruplex structures and functions, together with other analyt- ical methods, including various footprinting techniques. Ó 2010 Elsevier Ltd. All rights reserved. A wide variety of RNA plays an important role in the regulation of biological function within cells. The function of RNA largely de- pends on tertiary structures, in which RNA molecules use specific hydrogen bonds, including Watson–Crick and Hoogsteen bonds, to form active structures. Among a variety of RNA ternary folded structures, quadruplexes are one of the unique structures, in which guanine tetrads make Hoogsteen hydrogen bonds with each other in plane with incorporating metal ion, such as potassium and so- dium, inside the tetrads. 1–3 Recent reports have demonstrated that quadruplex structures in untranslated regions (UTRs) in mRNAs are involved in the translational regulation in vitro and vivo. 4–6 Computational predictions 7 together with various analytical methods, including spectroscopic methods 8,9 and footprinting techniques, 10–12 are commonly used to deduce secondary and ter- tiary structures of RNAs. Each technique has inherent advantages and disadvantages with regard to the quadruplex formations in RNAs, although spectroscopic analyses by using model oligonucle- otides suggested that guanine-rich RNAs are easy to form quadru- plex structures. 4–6,13 Simple and sensitive methods addressing RNA quadruplexes would provide deeper understandings of RNA quad- ruplex structures and functions. Reverse transcriptases have been used to predict stable second- ary structures in RNAs, involving quadruplex structures, however, the sensitivities of these assays are sometimes too low to fully ad- dress the sequence–structure relationships of quadruplex forma- tion. 14,15 Here, we reported a reverse transcription based method to precisely evaluate the stability and the structural diversity of quadruplexes in RNAs. In order to gain insight into local quadruplex structures on an RNA template, we studied an RNA-dependent DNA polymerase stop assay, named a reverse transcriptase stop assay (RTase stop assay) ( Fig. 1A). In the RTase stop assay, reverse transcriptases, such as those originated from Moloney murine leukemia virus (M-MuLV), proceeded along RNA templates until the enzyme encountered stable RNA quadruplex structures. Interference of RTase reaction by quadruplex structures on the templates would result in the production of truncated complement DNA products, which can be detected by PAGE analyses. 16,17 First, a human telomeric sequence 5 0 -r(UUAGGG) 4 UUA-3 0 (R- telo27) was chosen as a model of RNA quadruplexes. 13,18 R-telo27 showed UV-melting curves with an inverse transition at 295 nm. 19 The melting temperature showed a significant KCl concentration- dependency ( Fig. 1B) with T m values of 61.7 ± 0.2 °C and 76.5 ± 0.3 °C in 10 and 100 mM KCl, respectively. CD spectra of R-telo27 in the both KCl solutions suggested a parallel stranded quadruplex conformer, exhibiting a strong positive peak at 263 nm and a relatively weak peak near 240 nm ( Fig. 1C). These data suggested that R-telo27 having a four-telomeric-repeat affor- ded quadruplex structures with a different stability, depending on the KCl concentrations. With the biophysical information of quadruplex stabilities in hand, we next investigated quadruplex formations by the RTase stop assay on RNA templates that contained the R-telo27 sequence at the center of the template. In 100 mM KCl, the RTase reaction was exclusively interrupted at the first GGG site from 3 0 -end of the templates, where a plausible quadruplex was formed, regard- 0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2010.01.158 * Corresponding author. E-mail address: nakatani@sanken.osaka-u.ac.jp (K. Nakatani). Bioorganic & Medicinal Chemistry Letters 20 (2010) 2350–2353 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl