New size-expanded RNA nucleobase analogs: A detailed theoretical study

Highlights

• Four new size-expanded RNA nucleobase analogs are designed.

• The structural, electronic and optical properties are studied theoretically.

• The IPs and H–L gaps of the base analogs are smaller than those of natural ones.

• These analogs can form stable Watson–Crick base pairs with natural counterparts.

• All base analogs display visible emissions like their parental bases.

Abstract

Fluorescent nucleobase analogs have attracted much attention in recent years due to their potential applications in nucleic acids research. In this work, four new size-expanded RNA base analogs were computationally designed and their structural, electronic, and optical properties are investigated by means of DFT calculations. The results indicate that these analogs can form stable Watson–Crick base pairs with natural counterparts and they have smaller ionization potentials and HOMO–LUMO gaps than natural ones. Particularly, the electronic absorption spectra and fluorescent emission spectra are calculated. The calculated excitation maxima are greatly red-shifted compared with their parental and natural bases, allowing them to be selectively excited. In gas phase, the fluorescence from them would be expected to occur around 526, 489, 510, and 462 nm, respectively. The influences of water solution and base pairing on the relevant absorption spectra of these base analogs are also examined.

Introduction

As one of the most informative and sensitive analytical techniques, fluorescence spectroscopy plays key roles in modern research. Indeed, unraveling the inner workings of complex biomolecular assemblies relied on the development of fluorescence spectroscopic techniques. As we all know, natural nucleobases display extremely low fluorescent quantum yields and ultra-short excited-state lifetimes in both solution and gas phase. Therefore, the lack of naturally occurring fluorescent bases has triggered the development of unnatural nucleobase analogs with modified fluorescent properties that can be incorporated into oligonucleotides as quasi-intrinsic probes using standard automated synthetic methods [1], [2], [3]. In general, these fluorescent base analogs need to achieve isomorphicity with respect to natural ones in order to minimize structural and functional perturbations. A large number of nucleobase analog probes are now available, some even commercially, for incorporation into oligonucleotides for biophysical and biochemical studies [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. For example, the adenine analog 2-aminopurine has been used as a site-specific probe of nucleic acid structure and dynamics [4]. The cytosine analog, pyrrolocytosine, whose fluorescence yield is sensitive to the local structure of the biomolecules, is used as fluorescence probe in experimental studies of nucleic acid dynamics [5]. Hawkins and colleagues have described the synthesis of several pteridine base analogs [6], and some of which are highly fluorescent with Φ_F ranging from 0.03 to 0.88. Similarly, Saito and co-workers have produced a number of nucleobases with attractive fluorescent properties by fusing benzo- and naphtho-ring systems to hydrogen bonding aromatic rings [7]. Recently, Tor and co-workers synthesized four fluorescent thieno-modified base analogs, namely ^thA, ^thG, ^thU, and ^thC, which have been demonstrated to be faithful isomorphic nucleobase surrogates [8]. Their excited-state properties were theoretically studied by several groups [9], [10]. Samanta et al. investigated the electronic and optical properties of these newly designed bases and demonstrated that the thieno modification significantly modifies the electronic and optical properties, although the basic structural and bonding aspects remained the same [9]. Gedik and Brown investigated the absorption and fluorescence energies of these bases, and it was found that the TD-B3LYP method predicts energies closet to experimental values, while the TD-PBE0 method gives the best overall representation of the experimental findings [10].

An important strategy for designing fluorescent base analogs is size-expansion, which is achieved by fusing additional aromatic rings into the pyrimidine and purine scaffolds [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Having an extended aromatic surface typically results in favorable photophysical properties such as longer wavelength excitation as well as enhanced emission. To date, a lot of size-expanded base analogs have been designed and synthesized by using different aromatic rings including benzene [11], [12], [13], naphthalene [14], [15], [16], thiophene [17], [18], furan [19], pyrrole [18], [19], and other heterorings [19]. Experimentally, the first expanded nucleobase reported in the literature was designed by Leonard’s group, which is obtained by inserting a benzene spacer into natural adenine [11]. Along a similar line, Kool and co-workers have synthesized several variations of size-expanded base analogs known as x-bases [12], y-bases [13], and yy-bases [14]. The x- and y-bases are expanded by a benzene spacer and are ∼2.4 Å wider than natural ones, but differ in the direction of the expansion vector. The yy-bases were obtained by naphtho-homologation, and they have been paired with their natural counterparts to form a self-assembled yyDNA duplex that contain base pairs ∼4.8 Å wider than those in natural DNA. Thanks to the extra benzene/naphthalene ring, these size-expanded bases are fluorescent, a property making them good candidates for applications in nucleic acid research. Indeed, the x-bases have been tested in the arena of nucleic acid detection and it was found that in some cases these size-expanded bases could selectively recognize and report on DNA sequences and structures via their built-in fluorescence [12b]. Recently, the same group has synthesized a complete four-letter benzo-expanded RNA genetic set (xRNA) [12c]. It has been demonstrated that the xRNA nucleosides are efficient fluorophores [12c] with emission maxima of 369–411 nm. Similarly, Seley-Radtke and colleagues have synthesized several tricyclic purine analogs by inserting a thiophene ring into the purine scaffold [17]. These analogs have been demonstrated to have promising chemotherapeutic activity and their photophysical properties were theoretically investigated by the current group [18a]. Theoretically, Bu and co-workers designed a series of hetero-ring-expanded nucleobase analogs and studied their electronic spectroscopic properties [19]. Similarly, Liu et al. designed several size-expanded nucleobases and studied their absorption and emission spectra [20]. Clearly, these investigations have provided some useful information for design and synthesis of the unnatural fluorescent nucleobases.

One focus of our research has involved the design of structurally unique nucleobase analogs with enhanced electronic and fluorescent properties. In the present work, motivated by the proceeding success in designing and synthesizing size-expanded bases with enhanced fluorescent properties mentioned above, we design four benzo-expanded base analogs by inserting a benzene ring into the thieno-analogs synthesized recently by Tor’s group [8]. The newly designed RNA base analogs were named as xthA, xthG, xthC, and xthU, respectively (Fig. 1). Compared with their parental bases (termed as th-bases), these novel analogs still retain the main features, especially the Watson–Crick (WC) faces, and they are expected to retain the necessary hydrogen bonds to form the canonical WC pairs, while having enlarged base stacking interactions. Herein, we use theoretical methods to obtain the structural, electronic and optical properties of these base analogs and examine how the relevant properties are changed compared with their parental compounds. We hope the present study will be helpful for experimental design of novel expanded RNA/DNA and these novel base analogs could be new members to the big family of fluorescent base analogs.