Danith H. Ly

High Yield, Large Scale Synthesis of Thiolate-Protected Ag7 Clusters

We report a high yielding synthesis of truly monodisperse, thiolate-protected silver clusters via a rationally designed approach. The cluster composition was determined by electrospray ionization (ESI) mass spectrometry to be Ag(7)(DMSA)(4), where DMSA represents meso-2,3-dimercaptosuccinic acid. The Ag(7) thiolate clusters exhibit distinct optical properties. The approach developed in this work provides some insight into the cluster growth kinetics and may be extendable to the synthesis of other sized silver nanoclusters.

Synthesis of Conformationally Preorganized and Cell-Permeable Guanidine-Based γ-Peptide Nucleic Acids (γGPNAs)

A general method for preparing optically pure guanidine-based gamma-peptide nucleic acid (gammaGPNA) monomers for all four natural nucleobases (A, C, G, and T) is described. These second-generation gammaGPNAs differ from the first-generation GPNAs in that the guanidinium group is installed at the gamma- instead of the alpha-position of the N-(2-aminoethyl)glycine backbone unit. This positional switch enables GPNAs to be synthesized from relatively cheap L- as opposed to D-amino acids. Unlike their alpha-predecessors, which are randomly folded, gammaGPNAs prepared from L-amino acids are preorganized into a right-handed helix and bind to DNA and RNA with exceptionally high affinity and sequence selectivity and are readily taken up by mammalian cells.

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.

Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA

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.

Loop and Backbone Modifications of Peptide Nucleic Acid Improve G-Quadruplex Binding Selectivity

Targeting guanine (G) quadruplex structures is an exciting new strategy with potential for controlling gene expression and designing anticancer agents. Guanine-rich peptide nucleic acid (PNA) oligomers bind to homologous DNA and RNA to form hetero-G-quadruplexes but can also bind to complementary cytosine-rich sequences to form heteroduplexes. In this study, we incorporated backbone modifications into G-rich PNAs to improve the selectivity for quadruplex versus duplex formation. Incorporation of abasic sites as well as chiral modifications to the backbone were found to be effective strategies for improving selectivity as shown by UV-melting and surface plasmon resonance measurements. The enhanced selectivity is due primarily to decreased affinity for complementary sequences, since binding to the homologous DNA to form PNA-DNA heteroquadruplexes retains high affinity. The improved selectivity of these PNAs is an important step toward using PNAs for regulating gene expression by G-quadruplex formation.

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

Effect of Backbone Flexibility on Charge Transfer Rates in Peptide Nucleic Acid Duplexes

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.