Srinivas Rapireddy

Strand Invasion of Extended, Mixed-Sequence B-DNA by γPNAs

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 gammaPNA is required for invasion in this case, and no nucleobase substitution is needed.

Electronic Barcoding of a Viral Gene at the Single-Molecule Level

A new single-molecule approach for rapid and purely electronic discrimination among similar genes is presented. Combining solid-state nanopores and γ-modified synthetic peptide nucleic acid probes, we accurately barcode genes by counting the number of probes attached to each gene and measuring their relative spacing. We illustrate our method by sensing individual genes from two highly similar human immunodeficiency virus subtypes, demonstrating feasibility of a novel, single-molecule diagnostic platform for rapid pathogen classification.

Sequence-unrestricted, Watson-Crick recognition of double helical B-DNA by (R)-miniPEG-γPNAs.

Development of general principles for designing molecules to bind sequence specifically to double-stranded DNA (dsDNA) has been a long-sought goal of bioorganic chemistry and molecular biology. Pursuit of this goal, in the past, has generally been focused on the minor and major grooves—in large part, because of the ease of accessibility of the chemical groups that reside on these external parts of the double helix and the precedence for their recognition in nature. It was long recognized that while Watson–Crick (W–C) base-pairing provides a more direct and specific means for establishing sequence-specific interactions with nucleic acid biopolymers, such as DNA and RNA, it would be difficult to do so with intact double helical DNA because of the preexisting base pairs. This effort has so far led to the development of several major classes of antigene molecules, with the likes of triplexforming oligonucleotides, minor-groove binding polyamides, and major-groove binding zinc-finger peptides. While they can be designed to bind sequence specifically to dsDNA, there are still remaining issues with sequence selection, specificity and/or target length that have not yet been completely resolved, 13, 17–19] although some progress has been made in recent years. Over the past two decades, peptide nucleic acids (PNAs), a particular class of nucleic acid mimics comprised of a pseudopeptide backbone (Scheme 1 A), have been shown to be capable of invading dsDNA. This finding is significant because, contrary to the traditional belief, it demonstrates that the DNA double helix is relatively dynamic at physiological temperatures, and that W–C base-pairing interactions can be established with intact dsDNA. Though promising as antigene reagents, because of the specificity of recognition and generality in sequence design, PNA binding is presently limited to mostly homopurine and homopyrimidine targets. Mixed-sequence PNAs have been shown to be capable of invading topologically constrained supercoiled plasmid DNA, conformationally perturbed regions of genomic DNA and duplex termini ; however, they are unable to invade the interior regions of double helical B-form DNA (B-DNA)—the most stable form of DNA double helix. “Tail-clamp” 31] and “doubleduplex invasion” strategies have subsequently been developed and have enabled mixed-sequence PNAs to invade BDNA, but they are not without limitations. Recently we showed that mixed-sequence PNAs, when preorganized into a right-handed helix by installing an (S)-Me stereogenic center at the g-backbone (Scheme 1 B) can invade BDNA. However, all of our studies so far have been limited to a few selected sequences due to the poor water solubility and propensity of these first-generation gPNAs to aggregate and adhere to surfaces and other macromolecules, including DNA, in a nonspecific manner. This problem is exacerbated Scheme 1. Chemical structures of: A) PNA, B) l-alanine-derived gPNA (gPNA), and C) (R)-MiniPEG-containing gPNA (gPNA). See ref. [36] for the synthesis of gPNA monomers; the methyl ether protecting group of miniPEG side-chain is removed in the final cleavage/deprotection step of oligomer synthesis.

Strand Invasion of Mixed-Sequence, Double-Helical B-DNA by γ-Peptide Nucleic Acids Containing G-Clamp Nucleobases under Physiological Conditions

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.

A Simple Cytosine to G-Clamp Nucleobase Substitution Enables Chiral γ-PNAs to Invade Mixed-Sequence Double-Helical B-form DNA

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. [1] Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. [2] While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, [3–5] polyamides, [6–8] and zinc-finger-binding pep

RTD-1mimic containing γPNA scaffold exhibits broad-spectrum antibacterial activities.

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.

Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models

Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.

Effect of Steric Constraint at the γ-Backbone Position on the Conformations and Hybridization Properties of PNAs

Conformationally preorganized peptide nucleic acids (PNAs) have been synthesized through backbone modifications at the γ-position, where R = alanine, valine, isoleucine, and phenylalanine side chains. The effects of these side-chains on the conformations and hybridization properties of PNAs were determined using a combination of CD and UV-Vis spectroscopic techniques. Our results show that the γ-position can accommodate varying degrees of sterically hindered side-chains, reaffirming the bimodal function of PNAs as the true hybrids of “peptides” and “nucleic acids.”

Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. IMPORTANCE Bloodstream infections continue to be a major cause of death for hospitalized patients, despite significant improvements in both the availability of treatment options as well their application. Since early and targeted antimicrobial intervention is one of the prime determinants of patient outcome, the rapid identification of the pathogen can be lifesaving. Unfortunately, current diagnostic approaches for identifying these infections all rely on time-consuming blood culture, which precludes immediate intervention with a targeted antimicrobial. To address this, we have developed and characterized a new and comprehensive methodology, from patient specimen to result, for the rapid identification of both bacterial and fungal pathogens without the need for culturing. We anticipate broad interest in our work, given the novelty of our technical approach combined with an immense unmet need. Bloodstream infections continue to be a major cause of death for hospitalized patients, despite significant improvements in both the availability of treatment options as well their application. Since early and targeted antimicrobial intervention is one of the prime determinants of patient outcome, the rapid identification of the pathogen can be lifesaving. Unfortunately, current diagnostic approaches for identifying these infections all rely on time-consuming blood culture, which precludes immediate intervention with a targeted antimicrobial. To address this, we have developed and characterized a new and comprehensive methodology, from patient specimen to result, for the rapid identification of both bacterial and fungal pathogens without the need for culturing. We anticipate broad interest in our work, given the novelty of our technical approach combined with an immense unmet need.

Synthesis of optically pure γPNA monomers: a comparative study

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.