Author: Jonathan Sczepanski
Department of Chemistry
Texas A&M University
College Station, TX
When you think about DNA, do you consider its stereochemistry? You should! DNA is a chiral molecule, and consequently, has non-superimposable mirror images (enantiomers). The source of chirality in DNA is the deoxyribose sugar, which contains three chirality centers (Figure 1). On one “hand”, native DNA found in all living organisms consists exclusively of D-deoxyribose sugars (D-DNA) and forms right-handed helices. On the other hand, its enantiomer, L-DNA, consists of L-deoxyribose sugars and forms left-handed helices. L-DNA is not found in nature, but can be prepared synthetically in the laboratory, with the earliest syntheses of L-oligonucleotides dating back to the 1970s.1 As enantiomers, D-DNA and L-DNA are physically indistinguishable, except for their opposed optical activity, making them identical from a design perspective (i.e. same hybridization rates, duplex thermal stability, etc.). Importantly, L-DNA is orthogonal to the stereospecific environment of native biology, which has evolved to recognize D-nucleic acids. As a result, L-DNA is highly resistant to nuclease degradation and off-target interactions with other cellular macromolecules.2-3 These favorable properties, along with the recent commercialization of L-DNA phosphoramidite building blocks, has catalyzed the development of many promising L-DNA based biotechnologies.4
One of the first5-6, and still common, uses of L-DNA is in the form of aptamers. To date, L-DNA aptamers (also called Spiegelmers) have been evolved to bind a variety of targets, including small molecules, peptides, and proteins.7 Our laboratory has recently pioneered the use of L-DNA aptamers to bind native D-RNA structures.8 When applied in vivo, Spiegelmers retain a high affinity for their targets, while being nontoxic and have very low immunogenic potential.9 Given these desirable properties, it is not surprising that several Speigelmer therapeutics are in various stages of clinical development.10
Another powerful application of L-DNA is in the construction of bio-orthogonal molecular sensors. For example, L-DNA-based molecular beacons, which are not substrates for native polymerases, can be used for optical monitoring of PCR reactions, a method referred to as “adaptive PCR”.11 L-DNA molecular beacon probes have also been employed for measuring intracellular temperatures.12 DNA-based sensors for achiral analytes can be mirrored without a loss in activity. This property was exploited to generate nuclease-resistant L-DNAzyme sensors for monitoring metal ion concentrations in living cells.13-14 In the case of chiral analytes, use of both enantiomers of the corresponding nucleic acid sensor enables rapid screening of enantiomeric purity.15
Watson–Crick base pairing between complementary strands is stereospecific, and thus, L-DNA is incapable of hybridizing to native DNA and RNA.3, 16 While this property excludes the direct use of L-DNA as antisense agents, it ensures that sensors and other biotechnologies constructed from L-DNA have minimal off-target hybridization, an important consideration for analytical applications. Indeed, one of the earliest applications of L-DNA was the development of a universal microarray platform that employed L-DNA capture strands as a means to reduce cross-hybridization of nucleic acid samples with different regions of the array.3 The stereospecificity of hybridization can also be exploited to create internal controls during sample analysis.17
Although D-DNA and L-DNA do not interact directly, sequence information can still be transferred between the two enantiomers via strand-displacement strategies.18-20 For example, our laboratory showed that DNA enantiomers can be sequence-specifically interfaced via toehold-mediated strand-displacement from peptide nucleic acid (PNA), a process referred to as “heterochiral” strand displacement (Figure 2).18 Because PNA is achiral, and hybridizes equally well to both D-DNA and L-DNA, it functions as the intermediary, allowing a strand of D-DNA to displace a strand of L-DNA (and vice versa) in a sequence-specific manner. In principle, heterochiral strand displacement allows for any D-nucleic acid target (DNA or RNA) to be interfaced with sensors and nanodevices composed of bioorthogonal L-DNA. For example, we used this approach to interface microRNAs with L-DNA-based logic circuits and catalytic amplifiers in vitro18, 21-22, and with an L-RNA-based fluorescent biosensor in living cells.23 Chimeric strands of both D-DNA and L-DNA also enable native nucleic acids to be interfaced with L-DNA, and have been employed for various biosensing purposes.24-25
Figure 2. Schematic illustration of the heterochiral toehold-mediated strand displacement reaction. Nucleic acids are depicted as lines with half arrows denoting the 3’ end and an asterisk indicating complementarity between sequence domains. The single-stranded toehold domain (t*) resides on the achiral PNA strand in the L-DNA/PNA heteroduplex, allowing a D-Input strand bind (via t and t*) and displace the incumbent L-Output strand.
It is clear from the examples discussed herein (and elsewhere4) that L-DNA provides a powerful opportunity to develop nucleic acid-based technologies having capabilities not possible using only the native stereoisomer. As more researchers step “Through the Looking-Glass”, and use of mirror image DNA becomes more routine, completely new and exciting applications will become possible.