Placing Extra Components into RNA by Specific Transcription Using Unnatural Base Pair Systems
2006; Future Science Ltd; Volume: 40; Issue: 6 Linguagem: Inglês
10.2144/000112187
ISSN1940-9818
Autores Tópico(s)DNA and Nucleic Acid Chemistry
ResumoBioTechniquesVol. 40, No. 6 Techniques EssayOpen AccessPlacing Extra Components into RNA by Specific Transcription Using Unnatural Base Pair SystemsIchiro HiraoIchiro HiraoProtein Research Group, RIKEN Genomic Sciences Center, Kanagawa, JapanPublished Online:21 May 2018https://doi.org/10.2144/000112187AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionRNA molecules, which can be simply prepared and amplified by transcription using DNA templates, display versatile functionalities depending on their sequences and higher-order structures. This characteristic allows us to generate novel species of RNA that bind target molecules (aptamers) and catalysts (ribozymes) by in vitro selection methods using large populations of random RNA sequences. Over the last 15 years, researchers have created many aptamers and ribozymes for their purposes. Consequently, a modified RNA aptamer that binds to vascular endothelial growth factor was recently approved as a new treatment for age-related macular degeneration (1). In addition, efficient ribozymes for charging amino acid analogs onto a transfer RNA (tRNA) are available for incorporating amino acid analogs into proteins (2). As a result, RNA molecules have become useful biopolymers for biomedical research, diagnostics, and therapeutics.Despite these prospects, RNA-based biotechnology is still restricted. One of the problems is that RNA molecules comprise only four different, but similar, components (nucleotides). In contrast, proteins consist of 20 different, unique amino acids, including acidic, basic, and hydrophobic residues. In fact, in the evolution of life, the limited variety of nucleotides caused protein enzymes to displace most of the catalytic RNA functions, although various ribozymes might have appeared in the early stage of the beginning of life. This fact paradoxically suggests that even simple RNA molecules are capable of working as functional molecules and that their potential could be increased further by introducing extra components into RNA.Nucleotide analogs can be introduced into RNA by several methods, such as chemical synthesis, posttranscriptional modification, and enzymatic incorporation by transcription. Although chemical synthesis is useful for the site-specific introduction of chemically stable analogs (3), it is difficult to synthesize RNA molecules longer than 100 nucleotides (100-mer). Other methods, such as chemical modifications of RNA molecules and enzymatic incorporation during transcription, were also developed. For example, chemical biotinylation of RNA at its 3′ terminus was achieved by using biotin-conjugated hydrazide for the immobilization of RNA molecules, in which the 2′,3′-dihydroxy groups of the 3′-terminal nucleoside are oxidized to be functionalized (2,4,5). In addition, RNA can be biotinylated at the 5′ end by a one-step transcription procedure, using biotinyl-guanosine 5′-monophosphate under the control of the conventional T7 promoter (6) or N6-biotin derivatives of AMP as transcription initiators under the T7 Φ2.5 promoter (7). However, these methods are restricted to the terminal modification of RNA molecules. Alternative methods involve random incorporation of modified nucleotides by transcription (8), but it is obviously impossible to control the modification positions.A more attractive method is the expansion of the genetic alphabet by an unnatural base pair system (9), allowing for the site-specific, enzymatic incorporation of extra components by RNA polymerases, mediated by the unnatural base pair (Figure 1 A). For this purpose, an unnatural base pair that selectively and efficiently functions together with the natural A-T(U) and G-C base pairs in transcription is required. In this essay, we will discuss the unnatural base pair systems, focusing on our research, for the site-specific incorporation of nucleotide analogs into RNA molecules by transcription using T7 RNA polymerase, the most useful enzyme for RNA preparation.Figure 1. Specific transcription mediated by an unnatural base pair and unnatural base pairs that function in transcription.(A) Scheme for the site-specific incorporation of an extra base, N2, by T7 transcription using the unnatural N1-N2 pair, where N1 = isoC, x, s, orv, and N2 = isoG or y. (B)The Benner group's isoG-isoC and X-K pairs. (C)The Hirao group's x-y, s-y, and v-y pairs and the noncognate s-T pair.Creation of Unnatural Base PairsThe first unnatural base pair system was developed by Benner and colleagues. They designed and synthesized a series of unnatural base pairs, such as isoguanine-isocytosine (isoG-isoC) and xanthosine-diaminopyrimidine (X-K) (Figure 1B), with different hydrogen-bonding patterns from those of the natural base pairs (10,11). The A-T(U) and G-C base pairs have two and three hydrogen bonds, respectively. Each hydrogen bond is composed of the hydrogen-donor and hydrogen-acceptor residues or atoms, and the different combinations of donor-acceptor patterns in the unnatural base pairs from those in the natural base pairs confer their selectivity in DNA and RNA biosyntheses. The isoG-isoC pair was used for the introduction, by T7 RNA polymerase (12) of a modified isoG, N6-(6-aminohexyl)-isoG, into RNA fragments, using DNA templates containing isoC. The extra amino group of the modified isoG in the RNA fragments could be useful as a platform for post-transcriptional modification. Unfortunately, this unnatural base pair system has some shortcomings, including mispairing with the natural bases because of the tautomerization of isoG and the poor recognition of the 2-aminopyrimidines, such as isoC and K, by some RNA polymerases.Recently, we developed several unnatural bases, among which some pairing combinations can function in transcription as an extra base pair. To further improve the selectivity of Benner's base pairs, we combined the concepts of the hydrogen-bonding pattern and the shape complementarity by using steric effects, as suggested by Kool and colleagues (13,14). Then, we designed unnatural base pairs between 2-amino-6-dimethylaminopurine (x) and 2-oxopyridine (y) (15), 2-amino-6-(2-thienyl)purine (s) and y (16), and 2-amino-6-(2-thiazolyl)purine (v) and y (17) (see Figure 1C). In these unnatural base pairs, the bulky groups at position 6 of x, s, and v sterically clash with the natural bases and effectively prevent their noncognate pairings, but the relatively small hydrogen at position 6 of y maintains the shape complementarity of the pairings with x, s, or v. Consequently, the substrate of y (yTP) can be site-specifically incorporated into RNA, opposite x, s, or v in DNA templates, by T7 RNA polymerase. The DNA templates containing x, s, or v are prepared with a DNA synthesizer using their phosphoramidite derivatives. As compared with the bulky 6-dimethylamino group of x, the 6-heterocycles of s and v more effectively prevent the noncognate pairings, and moreover, duplex DNAs containing the s-y or v-y pair display higher thermal stability than those containing the x-y pair. Thus, the selectivity of the s-y and v-y pairings is higher than that of the x-y pairing in T7 transcription. This specific transcription using the s-y pair can also be combined with an in vitro translation system for incorporating amino acid analogs into proteins by expansion of the genetic code (16). In the coupled transcription-translation system, a messenger RNA (mRNA) including an extra codon, yAG, was transcribed from its s-containing DNA template, and then, human Ras protein, in which an amino acid analog, 3-chlorotyrosine, was site-specifically incorporated at position 32, was synthesized by using the 3-chlorotyrosine-charged tRNA containing the anticodon CUs.Functional RNA Molecules with Extra ComponentsAs for creating RNA molecules with functions other than coding, the y base acts only as a uracil analog. It lacks the 4-keto group of U, and thus, we chemically synthesized a series of modified y-substrates, in which several functional groups were added to position 5 of y. These modified y-substrates, illustrated in Figure 2, can be prepared by the nucleoside of 5-iodo-y, which is obtained from the y nucleoside by iodination using N-iodosuccinimide (18). All of the substrates can be site-specifically incorporated into RNA opposite s or v in DNA templates by standard T7 transcription.Figure 2. A series of functional substrates of y capable of site-specific incorporation into RNA by specific transcription.The iodo-y (I-y) base is a photosensitive component that is capable of cross-linking by irradiation at 312 nm (19) with nearby reactive residues in a target molecule. In addition, RNA molecules containing I-y at a specific position could be useful as a rational phasing tool for X-ray crystallography. The phenylethynyl residue (Ph-y) in RNA molecules stabilizes RNA tertiary structures by its extended stacking ability with neighboring bases (20). Introducing an aminohexanamide-1-pro-pynyl residue (NH2-hx-y) into a specific position of transcripts enables the site-specific posttranscriptional modification of the RNA molecules (18). Biotinylated-y (Bio-y) is used for the site-specific biotinylation of RNA transcripts to facilitate the immobilization of RNA molecules (21). Fluorophore-linked y bases, such as FAM (FAM-y and FAM-hx-y), TAMRA (TAMRA-hx-y), and dansyl (Dansyl-y), are used for site-specific fluorescent labeling of RNA molecules (18).The incorporation sites of the functional y bases in RNA molecules are designed and located in three main ways: (i) simple labeling of the terminal positions of RNA, (ii) rational design of the incorporation sites, and (iii) selection methods from a combinatorial pool containing extra random bases.First, for simple fluorescent labeling or immobilization of RNA molecules, the fluorescent or biotin-linked y bases should be incorporated into sites that are not related to the RNA activity. In this regard, the 3′ region of the RNA molecules, especially those obtained by in vitro selection, is suitable, because these RNA molecules contain 5′ and 3′ constant regions. The constant regions are also useful for preparing and amplifying the DNA templates containing s or v by PCR with s- or v-containing 3′ primers. By using FAM-hx-yTP or Bio-yTP, we performed the fluorescent labeling and immobilization of an anti-Raf-1 RNA aptamer (100-mer) that binds to the human Raf-1 protein and inhibits the interaction between Raf-1 and Ras (22) (Figure 3A). The RNA aptamer was fluorescently labeled at position 90 without any significant activity loss; the FAM-labeled aptamer exhibited a binding constant (Kd = 184 ± 46 nM) comparable to that of the unmodified aptamer (Kd = 152 ± 23 nM) (18). In addition, the biotinylated RNA aptamer was efficiently immobilized onto sensor chips and detected the Raf-1 protein (21).Figure 3. Site-specific fluorescent labeling and biotinylation of RNA molecules.(A) Preparation and amplification of the DNA template containing s and fluorescent labeling and biotinylation of the 3′ region of the anti-(Raf-1) RNA aptamer. (B) Site-specific FAM-y incorporation at position 6, in place of U6, in the theophylline binding aptamer. The aptamer was excited at 493 nm, and the fluorescent intensity was detected at 522 nm. Theophylline binding to the aptamer caused the fluorescent intensity to increase.Second, the incorporation sites for the functional y bases can be determined on the basis of the structural information of RNA molecules. This rational design is expected to endow the RNA molecules with novel functionality. For example, the specific fluorescent labeling of RNA aptamers can be used as a detection system for target molecules. Most RNA aptamers undergo structural changes upon binding to target molecules, and thus, by the introduction of fluorophores at specific positions within the aptamers, the conformational changes could be detected by alterations in the fluorescent intensity. By T7 transcription using FAM-yTP and a chemically synthesized s-containing template, we incorporated FAM-y in a specific position of a theophylline binding aptamer (23), on the basis of the higher-order structure of the aptamer (18) (Figure 3B). Upon binding to theophylline, the aptamer forms a unique base triplet, U6-U23-A28, next to the binding site (24). Since the 4-keto group of U6 does not involve the base triplet, we replaced U6 with FAM-y. Due to its hydrophobicity, the FAM residue resides inside the aptamer structure in the absence of theophylline, and its fluorescent intensity is reduced by stacking with neighboring bases. Upon theophylline binding, the FAM residue moves outside, and the fluorescent intensity increases. In contrast, 20 µM caffeine, an analog of theophylline, did not affect the fluorescent intensity. This sensor aptamer was able to selectively detect about 500 nM theophylline.Third, the combinatorial selection methods using RNA pools randomly containing unnatural bases are also a powerful tool for creating novel functional RNA molecules. However, there have been no reports on applying unnatural base pair systems to the selection methods. In vitro selection involves three steps: (i) PCR amplification of DNA templates, (ii) transcription to RNA molecules, and (iii) reverse transcription of the selected RNA molecules. Thus, to expand the genetic alphabet in the RNA pool, we need to develop an unnatural base pair system that functions in PCR, transcription, and reverse transcription. At present, several unnatural base pairs have been synthesized and tested in replication (25–30), and thus, in the near future, in vitro selection with the expanded genetic alphabet is likely to be realized. The most feasible selection method will be by using a pool of RNA containing an unnatural base flanked by two random regions. In accordance with the intended selection, a specific functional group can be attached to the unnatural base. For example, by adding a photo-reactive group to the extra base, researchers could select RNA aptamers that efficiently cross-link to a target molecule.Here, we have summarized the unnatural base pair systems for creating functional RNA molecules. Although this technology is still under investigation, some unnatural base pairs have been practically used for the site-specific incorporation of novel building blocks into RNA molecules. Extra functions have been added to RNA aptamers and ribozymes to increase their utility. We plan to pursue the potential of this technology by expanding its applications and improving the systems. 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