Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

The development of new RNA‐binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex‐forming peptide nucleic acids (PNAs) by further increasing the pKa of 2‐aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron‐donating 4‐methoxy substituent resulted in slight destabilization of the PNA–dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M‐modified PNA–dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4‐methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen‐bonding interactions that might be responsible for the high affinity and unusual RNA‐binding preference of PNAs.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

The successo fR NA interference (RNAi)a saresearch tool and potential therapeutic approachh as reinvigorated interesti nc hemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amidesm ay be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) criticalf or in vivo applications.I nt his study,w ei ntroduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutivea mide linkages retained high RNAi activity when paired with ap assenger strandhavingo ne amide modifica-tion between its first and second nucleosidesa tthe 5'-end. Further increasei nt he number of modifications decreased the RNAia ctivity;h owever,s iRNAs with sixa nd seven amide linkages still showedu seful target silencing. While an siRNA duplexh aving nine amide linkages retained somes ilencing activity,t he partial reduction of the negative charged id not enable passiveu ptake in HeLa cells. Our resultss uggest that further chemical modifications, in addition to amide linkages, are neededt oe nablec ellular uptake of siRNAs in the absence of transfection agents.[a] Dr.Scheme1.Synthesis of monomers 3 and 4.Steps:a)TOM-Cl, Bu 2 SnCl 2 , DIPEA,C lCH 2 CH 2 Cl, reflux,1h, 26 %o f5 and 41 %o f6;b)H 2 S( gas), pyridine/water(4:1), RT,overnight;c )p-methoxytrityl chloride,D MAP,pyridine, RT,overnight, 65 %o f7 and 65 %o f8;d)succinic anhydride, DBU, CH 2 Cl 2 , RT,30min, 80 %; e) P(OCH 2 CH 2 CN)(N(iPr) 2 ) 2 ,t etrazole, N-methylimidazole, CH 2 Cl 2 ,RT, 24 h, 35 %.

RNA-based technologies to control gene expression, such as, RNA interference (RNAi) and CRISPR-Cas9 have become powerful tools in molecular biology and genomics. The exciting potential that RNAi and CRISPR-Cas9 may also become new therapeutic approaches has reinvigorated interest in chemically modifying RNA to improve its properties for in vivo applications. Chemical modifications can improve enzymatic stability, in vivo delivery, cellular uptake, and sequence specificity; as well as minimize off-target activity of short interfering RNAs (siRNAs) and CRISPR associated RNAs. While numerous good solutions for improving stability towards enzymatic degradation have emerged, optimization of the latter functional properties remains challenging. In this Account, we discuss synthesis, structure, and biological activity of novel non-ionic analogues of RNA that have the phosphodiester backbone replaced by amide linkages (AM1). Our long-term goal is to use the amide backbone to improve the stability and specificity of siRNAs and other functional RNAs. Our work in this area was motivated by early discoveries that non-ionic backbone modifications, including AM1, did not disturb the overall structure or thermal stability of RNA duplexes. We hypothesized that the reduced negative charge and hydrophobic nature of the AM1 backbone modification might be useful in optimizing functional applications through enhanced cellular uptake, and might suppress unwanted off-target effects of siRNAs. NMR and X-ray crystallography studies showed that AM1 was an excellent mimic of phosphodiester linkages in RNA. The local conformational changes caused by the amide linkages were easily accommodated by small adjustments in RNA's conformation. Further, the amide carbonyl group assumed an orientation that is similar to one of the non-bridging P-O bonds, which may enable amide/phosphate mimicry by conserving hydrogen bonding interactions. The crystal structure of a short amide-modified DNA-RNA hybrid in complex with RNase H indicated that the amide N-H could also act as an H-bond donor to stabilize RNA-protein interactions; which is an interaction mode not available to phosphate groups. Functional assays established that amides were well tolerated at internal positions in both strands of siRNAs. Surprisingly, amide modifications in the middle of the guide strand and at the 5′-end of the passenger strand increased RNAi activity compared to unmodified siRNA. Most importantly, an amide linkage between the