Toward a General Approach for RNA-Templated Hierarchical Assembly of Split-Proteins

Abstract

IntroductionThe ability to conditionally elicit a response in the presence of a user-defined macromolecular target has great potential in biosensor design, targeted therapeutics, and a variety of applications in synthetic biology. With our enhanced understanding of the human genome, both DNA and RNA have become increasingly important biological targets related to diverse human disease. 1 General methods for nucleic acid targeting now include several hybridization based approaches such as RNA interference (RNAi), triplex-forming oligonucleotides (TFOs), peptide nucleic acids (PNAs), as well as the elegantly designed polyamides. 2 Nucleic acid detection most commonly relies on fluorescently labeled oligonucleotides, as in the case of fluorescence in situ hybridization (FISH). 3 However, the constitutive fluorescent signal associated with this class of probes necessitates washing steps and results in reduced sensitivity, which potentially limits the utility of this technique for in vivo imaging. 4 To address these concerns, recent efforts have been directed toward generating "turn-on" fluorescent or electrochemical sensors, which couple conditional signal output to target binding. 5 In a complementary approach, there has been recent interest in DNA-and RNA-templated reactions, wherein probe localization on a single-stranded nucleic acid target enables the specific chemical transformation of attached moieties. 6 Examples of nucleic acid-templated chemical reactions include FRET-and quencher-based autoligation probes, metallosalen−DNA conjugates and deoxyribozymes for DNA hydrolysis, and catalytically released cytotoxic drugs. 7 Additionally, there is great interest in the design of DNA-directed gain of function proteins, which cannot only serve as sensors and genome modifying agents, but also have potential as specific therapeutics. Many groups have described protein-based approaches for the conditional reassembly of fragmented proteins on double-stranded DNA (dsDNA) mainly utilizing the Cys 2 His 2 class of zinc fingers (ZFs) as targeting domains. 8, 9 Published in Journal of the American Chemical Society 132:33 (2010) Corresponding author -I. Ghosh, ghosh@email.arizona.edu AbstractThe ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ss-RNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3′ overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expa...

Turn-On DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA

“…Considering that closer spacing (0–1 bp) may preclude simultaneous binding of the detection domains, 36 the wide range of accessible target lengths is presumably attributable to the length and flexible constitution of the linkers connecting the detection domains to split luciferase. 42 To confirm this result and to investigate reassembly constraints associated with 8-oxoG detection, we next evaluated our DNA oxidation sensor using the same set of methylated oligonucleotides, which were exposed to oxidizing conditions of 1 mM H 2 O 2 and 30 μM CuCl 2 . Using this sensor pair, the distance between the single methylation site and the oxidized guanine base is not explicitly known.…”

“…However, since the absolute signal associated with the CLuciferase-OGG1 + MBD1-NLuciferase pair was 4.5-fold higher, this sensor architecture was chosen for further optimization. Because the linker joining the protein fusion may also affect the efficiency of reassembly, 42 a third OGG1 fusion construct (CLuciferase-33AA-OGG1) was generated that encodes a 33 AA linker for comparison to the original construct containing an 18 AA linker (Figure 3C). We produced each complementary set of biosensors, CLuciferase-18AA-OGG1 with MBD1-NLuciferase and CLuciferase-33AA-OGG1 with MBD1-NLuciferase, in a cell-free translation system, followed by addition of 7.1 nM of oxidized or non-oxidized methylated oligonucleotide.…”

“…36–38 Of the many signaling proteins that have been validated for use in split-protein reassembly strategies, we have recently established that the low background and high luminescent signal output of split firefly luciferase makes it particularly attractive for use in a cell-free translation system for biosensor generation. 39–42 In order to rapidly probe genome-wide DNA damage utilizing our cell-free split-protein methodology, we reasoned that we would require a generic localization domain for targeting our sensors to any damage-accessible DNA. Transcription factors are generally not suitable for this function, since binding should be largely independent of surrounding sequence.…”

Labeling native bacterial RNA in live cells

“…Protein mutagenesis is a time-consuming process and it limits application of this approach. Another method that used RNA-templated protein complementation has been developed but used for in vitro RNA detection only [3]. Therefore, detection of unmodified RNAs in vivo remains a daunting problem.…”

“…These results underscore the limitations of hybridization methods that use probes with constitutive fluorescent signal, which require washing steps to reveal the specific signal. These limitations of the pre-labeled probes have motivated the efforts to develop molecular sensors that display signal in the presence of the analyte only [3].…”