Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host’s genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

We report a systematic study examining two synthetic routes, reductive amination and Mitsunobu coupling, for preparation of chiral γ-peptide nucleic acid (γPNA) monomers and oligomers. We found that the reductive amination route is prone to epimerization, even under mild experimental conditions. The extent of epimerization could be minimized by utilizing a bulky protecting group such as PhFl; however, it is difficult to remove in the subsequent oligomer synthesis stage. On the other hand, we found that the Mitsunobu route produced optically superior products using standard carbamate protecting groups.

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities and high chemical, thermal, and enzymatic stability. However, for some, because of their intricate spatial arrangement and elaborate interstrand cross-linkages, they are difficult to prepare de novo in large quantities and high purity, due to the nonselective nature of disulfide-bond formation. We show that the disulfide bridges of RTD-1, a member of the θ-defensin subfamily, could be replaced with noncovalent Watson–Crick hydrogen bonds without significantly affecting its biological activities. The work provides a general strategy for engineering conformationally rigid, cyclic peptides without the need for disulfide-bond reinforcement.

Peptide nucleic acids (PNAs) make up the only class of nucleic acid mimics developed to date that has been shown to be capable of invading double-helical B-form DNA. Recently, we showed that sequence limitation associated with PNA recognition can be relaxed by utilizing conformationally preorganized γ-peptide nucleic acids (γPNAs). However, like all the previous studies, with the exception of triplex binding, DNA strand invasion was performed at relatively low salt concentrations. When physiological ionic strengths were used, little to no binding was observed. On the basis of this finding, it was not clear whether the lack of binding is due to the lack of base pair opening or the lack of binding free energy, either of which would result in no productive binding. In this work, we show that it is the latter. Under simulated physiological conditions, the DNA double helix is sufficiently dynamic to permit strand invasion by the designer oligonucleotide molecules provided that the required binding free energy can be met. This finding has important implications for the design oligonucleotides for recognition of B-DNA via direct Watson–Crick base pairing.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol (“miniPEG”, R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure R-MPγPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, R-MPγPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

We have determined the structure of a PNA−DNA duplex to 1.7 Å resolution by multiple-wavelength anomalous diffraction phasing method on a zinc derivative. This structure represents the first high-resolution 3D view of a hybrid duplex containing a contiguous chiral PNA strand with complete γ-backbone modification (“γPNA”). Unlike the achiral counterpart, which adopts a random-fold, this particular γPNA is already preorganized into a right-handed helix as a single strand. The new structure illustrates the unique characteristics of this modified PNA, possessing conformational flexibility while maintaining sufficient structural integrity to ultimately adopt the preferred P-helical conformation upon hybridization with DNA. The unusual structural adaptability found in the γPNA strand is crucial for enabling the accommodation of backbone modifications while constraining conformational states. In conjunction with NMR analysis characterizing the structures and substructures of the individual building blocks, these results provide unprecedented insights into how this new class of chiral γPNA is preorganized and stabilized, before and after hybridization with a cDNA strand. Such knowledge is crucial for the future design and development of PNA for applications in biology, biotechnology, and medicine.

This paper presents the results of an NMR spectroscopy and distance-restrained molecular dynamics (MD) study of a γ-methylated, palindromic, 8-base pair peptide nucleic acid (γ-PNA) duplex. The goal of this study was to examine the impact of the γ-backbone modification on the structure of the PNA duplex. The 2D NMR information involving the backbone methyl group, especially the NOEs between the methyl protons and those of the amide and methylene groups of the backbone, led to distance restraints useful in the elucidation of the structure of the backbone of γ-PNA. Integration of the NOE peaks resulted in 138 inter-proton distance restraints, which were used in ten independent simulated annealing followed by 2 ns restrained MD runs. These simulations led to the conclusion that the γ-PNA duplex adopts a general P-form helical structure similar to that observed for non-modified PNA but with a smaller base pair rise, which is an A-like helical feature, and a slight helical bending towards the major groove (PDB ID 2KVJ). These properties of the γ-PNA duplex may be induced by the γ-methyl group. A similar effect of the methyl group was revealed by a previous NMR study of single stranded γ-PNA [A. Dragulescu-Andrasi, S. Rapireddy, B. M. Frezza, C. Gayathri, R. R. Gil and D. H. Ly, J. Am. Chem. Soc., 2006, 128, 10258–10267]. It appears that the steric constraint exerted by the γ-methyl on the backbone orientation is relatively independent of the base pairing and stacking and thus is likely to manifest when other substituents are introduced at the γ-position of the PNA.

In this communication, we show that peptide nucleic acids (PNAs) with lengths of 15−20 nucleotides, when preorganized into a right-handed helix, can invade mixed-sequence double-helical B-form DNA (B-DNA). Strand invasion occurs in a highly sequence-specific manner through direct Watson−Crick base pairing. Unlike the previously developed double-duplex invasion strategy, which requires simultaneous binding of two strands of pseudocomplementary PNAs to DNA, only a single strand of γPNA is required for invasion in this case, and no nucleobase substitution is needed.

Guanidine-based peptide nucleic acid (GPNA) monomers and oligomers containing all four natural (adenine (A), cytosine (C), guanine (G), and thymine (T)) and two unnatural (2-thiouracil (sU) and 2,6-diaminopurine (D)) nucleobases have been synthesized. Thermal denaturation study showed that GPNA oligomers containing alternate D-backbone configuration bind sequence-specifically to DNA and, when incubated with mammalian cells, localized specifically to the endoplasmic reticulum (ER).

Guanidine-based peptide nucleic acid (GPNA) with a D-backbone configuration and alternate spacing binds sequence-specifically to RNA and is readily taken up by both human somatic and embryonic stem (ES) cells.