Bilingual Peptide Nucleic Acids: Encoding the Languages of Nucleic Acids and Proteins in a Single Self-Assembling Biopolymer.

Nucleic acids and proteins are the fundamental biopolymers that support all life on Earth. Nucleic acids store large amounts of information in nucleobase sequences while peptides and proteins utilize diverse amino acid functional groups to adopt complex structures and perform wide-ranging activities. Although nature has evolved machinery to read the nucleic acid code and translate it into amino acid code, the extant biopolymers are restricted to encoding amino acid or nucleotide sequences separately, limiting their potential applications in medicine and biotechnology. Here we describe the design, synthesis, and stimuli-responsive assembly behavior of a bilingual biopolymer that integrates both amino acid and nucleobase sequences into a single peptide nucleic acid (PNA) scaffold to enable tunable storage and retrieval of tertiary structural behavior and programmable molecular recognition capabilities. Incorporation of a defined sequence of amino acid side-chains along the PNA backbone yields amphiphiles having a "protein code" that directs self-assembly into micellar architectures in aqueous conditions. However, these amphiphiles also carry a "nucleotide code" such that subsequent introduction of a complementary RNA strand induces a sequence-specific disruption of assemblies through hybridization. Together, these properties establish bilingual PNA as a powerful biopolymer that combines two information systems to harness structural responsiveness and sequence recognition. The PNA scaffold and our synthetic system are highly generalizable, enabling fabrication of a wide array of user-defined peptide and nucleotide sequence combinations for diverse future biomedical and nanotechnology applications.

Elucidating Sequence-Assembly Relationships for Bilingual PNA Biopolymers.

Nucleic acids and proteins possess encoded "languages" that can be used for information storage or to direct function. However, each biopolymer is limited to encoding its respective "language." Using a peptide nucleic acid (PNA) scaffold, nucleobase and amino acid residues can be installed on a singular backbone, enabling a single biopolymer to encode both languages. Our laboratory previously reported the development of a "bilingual" PNA biopolymer that incorporates a sequence-specific nucleic acid code interspersed with hydrophobic (alanine) and hydrophilic (lysine) amino acid residues at defined positions to produce amphiphilic character. We observed the amphiphilic amino acid residues directing the biopolymer to undergo self-assembly into micelle-like structures, while the nucleic acid recognition was harnessed for disassembly. Herein, we report a series of bilingual PNA sequences having amino acid residues with varying lengths, functional group charges, hydrophobicities, and spacings to elucidate the effect of these parameters on micelle assembly and nucleic acid recognition. Negative charges in the hydrophilic block or increased bulkiness of the hydrophobic side chains led to assembly into similarly sized micelles; however, the negative charge additionally led to increased critical micelle concentration. Upon PNA sequence truncation to decrease the spacing between side chains, the biopolymers remained capable of self-assembling but formed smaller structures. Characterization of disassembly revealed that each variant retained sequence recognition capabilities and stimuli-responsive disassembly. Together, these data show that the amino acid and nucleic acid sequences of amphiphilic bilingual biopolymers can be customized to finely tune the assembly and disassembly properties, which has implications for applications such as the encapsulation and delivery of cargo for therapeutics.

Forced Intercalation Peptide Nucleic Acid Probes for the Detection of an Adenosine-to-Inosine Modification.

The deamination of adenosine to inosine is an important modification in nucleic acids that functionally recodes the identity of the nucleobase to a guanosine. Current methods to analyze and detect this single nucleotide change, such as sequencing and PCR, typically require time-consuming or costly procedures. Alternatively, fluorescent "turn-on" probes that result in signal enhancement in the presence of target are useful tools for real-time detection and monitoring of nucleic acid modification. Here we describe forced-intercalation PNA (FIT-PNA) probes that are designed to bind to inosine-containing nucleic acids and use thiazole orange (TO), 4-dimethylamino-naphthalimide (4DMN), and malachite green (MG) fluorogenic dyes to detect A-to-I editing events. We show that incorporation of the dye as a surrogate base negatively affects the duplex stability but does not abolish binding to targets. We then determined that the identity of the adjacent nucleobase and temperature affect the overall signal and fluorescence enhancement in the presence of inosine, achieving an 11-fold increase, with a limit of detection (LOD) of 30 pM. We determine that TO and 4DMN probes are viable candidates to enable selective inosine detection for biological applications.

Stimuli-responsive assembly of bilingual peptide nucleic acids.

Peptide nucleic acids (PNAs) are high-affinity synthetic nucleic acid analogs capable of hybridization with native nucleic acids. PNAs synthesized having amino acid sidechains installed at the γ-position along the backbone provide a template for a single biopolymer to simultaneously encode nucleic acid and amino acid sequences. Previously, we reported the development of "bilingual" PNAs through the synthesis of an amphiphilic sequence featuring separate blocks of hydrophobic and hydrophilic amino acid functional groups. These PNAs combined the sequence-specific binding activity of nucleic acids with the structural organization properties of peptides. Like other amphiphilic compounds, these γ-PNAs were observed to assemble spontaneously into micelle-like nanostructures in aqueous solutions and disassembly was induced through hybridization to a complementary sequence. Here, we explore whether assembly of these bilingual PNAs is possible by harnessing the nucleic acid code. Specifically, we designed an amphiphile-masking duplex system in which spontaneous amphiphile assembly is prevented through hybridization to a nucleic acid masking sequence. We show that the amphiphile is displaced upon introduction of a releasing sequence complementary to the masking sequence through toehold mediated displacement. Upon release, we observe that the amphiphile proceeds to assemble in a fashion consistent with our previously reported structures. Our approach represents a novel method for controlled stimuli-responsive assembly of PNA-based nanostructures.

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.