Design and enhanced gene silencing activity of spherical 2'-fluoroarabinose nucleic acids (FANA-SNAs).

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs.

Effect of 2'-5'/3'-5' phosphodiester linkage heterogeneity on RNA interference.

We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2.

Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of siRNA Duplexes.

In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting.

Minimalist Design of a Stimuli-Responsive Spherical Nucleic Acid for Conditional Delivery of Oligonucleotide Therapeutics.

In this work, we report a component-minimal spherical nucleic acid (SNA) from monodisperse DNA-polymer conjugates that can load and release nucleic acid therapeutics in a stimuli-responsive manner. We show that this vehicle assembles from only four strands, and conditional release of its antisense therapeutic cargo can be induced upon recognition of specific oligonucleotide triggers via strand displacement. The latter (triggers) may be a microRNA that offers additional synergistic therapy, in addition to the previously shown ability of the SNA to load hydrophobic drugs.

Templated synthesis of spherical RNA nanoparticles with gene silencing activity.

RNA has inherent therapeutic and structural properties that make it an important component of biologically-functional nanoparticles. Using DNA-amphiphiles as synthetic templates, we report the synthesis of two classes of RNA-amphiphiles that self-assemble into spherical nanoparticles in aqueous solution and show gene silencing activity.

Cellular Studies of an Aminoglycoside Potentiator Reveal a New Inhibitor of Aminoglycoside Resistance.

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has, however, dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators that work by inhibiting aminoglycoside N-6'-acetyltransferase, one of the most common determinants of aminoglycoside resistance.

Structural Studies and Gene Silencing Activity of siRNAs Containing Cationic Phosphoramidate Linkages.

A series of siRNA duplexes containing cationic non-bridging 3',5'-linked phosphoramidate (PN) linkages was designed and synthesized using a combination of phosphoramidite and H-phosphonate chemistries. Modified oligonucleotides were assayed for their thermal stability, helical structure, and ability to modulate the expression of firefly luciferase. We demonstrate that PN modifications of siRNAs are, in general, minimally destabilizing with respect to duplex thermal stability; destabilization can be mitigated through the incorporation of 2'-modified RNA-like residues or PN conjugates containing ionizable pendant moieties.

Efficient and Rapid Mechanochemical Assembly of Platinum(II) Squares for Guanine Quadruplex Targeting.

We present a rapid and efficient method to generate a family of platinum supramolecular square complexes, including previously inaccessible targets, through the use of ball milling mechanochemistry. This one-pot, two-step process occurs in minutes and enables the synthesis of the squares [Pt(en)(N∩N)][CFSO] (en= ethylenediamine, N∩N = 4,4'-bipyridine derivatives) from commercially available precursor KPtCl in good to excellent yields. In contrast, solution-based assembly requires heating the reagents for weeks and gives lower yields.

Precision spherical nucleic acids for delivery of anticancer drugs.

We report a spherical nucleic acid (SNA) system for the delivery of BKM120, an anticancer drug for treatment of chronic lymphocytic leukemia (CLL). While promising for cancer treatment, this drug crosses the blood-brain barrier causing significant side-effects in patients. The DNA nanoparticle encapsulates BKM120 in high efficiency, and is unparalleled in its monodispersity, ease of synthesis and stability in different biological media and in serum.

4'-C-Methoxy-2'-deoxy-2'-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing.

We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'β) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2'-F U. H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects.

Correction: Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown.

Correction for 'Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown' by Johans J. Fakhoury, et al., Nanoscale, 2015, 7, 20625-20634.

Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown.

Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells.

Locked 2'-Deoxy-2',4'-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity.

2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes.

An orthogonal photolabile linker for the complete "on-support" synthesis/fast deprotection/hybridization of RNA.

The preparation of a polystyrene solid support decorated with a photolabile linker is described. The entire post-synthetic processing of RNA can be carried out in the solid phase in a minimum amount of time. The deprotected RNA is available for "on-support" hybridization and photolysis releases siRNA duplexes under mild, neutral conditions.

Arabinonucleic acids: 2'-stereoisomeric modulators of siRNA activity.

We have investigated, for the first time, short interfering duplexes containing arabinonucleotides (ANA; the 2'-stereoisomer of RNA), as well as combinations of ANA with RNA, and their 2'-fluorinated derivatives 2F-ANA and/or 2'F-RNA. The results show that ANA is especially well accommodated in the sense strand of small interfering RNA (siRNA) duplexes, which can be extensively modified with little effect on potency. Furthermore, combining ANA with RNA and 2'F-ANA in siRNA passenger strands, particularly in patterns that bias duplex thermal stability, produces duplexes with similar (and sometimes enhanced) potency compared with native siRNA.

Development and characterization of gene silencing DNA cages.

RNA interference (RNAi) is a powerful therapeutic strategy that induces gene silencing by targeting disease-causing mRNA and can lead to their removal through degradation pathways. The potential of RNAi is especially relevant in cancer therapy, as it can be designed to regulate the expression of genes involved in all stages of tumor development (initiation, growth, and metastasis). We have generated gene silencing 3D DNA prisms that integrate antisense oligonucleotide therapeutics at 1, 2, 4, and 6 positions.

A platinum(II) phenylphenanthroimidazole with an extended side-chain exhibits slow dissociation from a c-Kit G-quadruplex motif.

A series of three platinum(II) phenanthroimidazoles each containing a protonable side-chain appended from the phenyl moiety through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) were evaluated for their capacities to bind to human telomere, c-Myc, and c-Kit derived G-quadruplexes. The side-chain has been optimized to enable a multivalent binding mode to G-quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high-throughput fluorescence intercalator displacement (HT-FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side-chains and other reported G-quadruplex binders.

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells.

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT).

Rolling circle amplification-templated DNA nanotubes show increased stability and cell penetration ability.

DNA nanotubes hold promise as scaffolds for protein organization, as templates of nanowires and photonic systems, and as drug delivery vehicles. We present a new DNA-economic strategy for the construction of DNA nanotubes with a backbone produced by rolling circle amplification (RCA), which results in increased stability and templated length. These nanotubes are more resistant to nuclease degradation, capable of entering human cervical cancer (HeLa) cells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation and release behavior.

Platinum(II) phenanthroimidazoles for targeting telomeric G-quadruplexes.

A rationally designed progression of phenanthroimidazole platinum(II) complexes were examined for their ability to target telomere-derived intramolecular G-quadruplex DNA. Through the use of circular dichroism, fluorescence displacement assays, and molecular modeling we show that these complexes template and stabilize G-quadruplexes from sequences based on the human telomeric repeat (TTAGGG)(n). The greatest stabilization was observed for the p-chlorophenyl derivative 6((G4)DC(50) =0.

Telomeric function of mammalian telomerases at short telomeres.

Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) known as TERT and an RNA known as TR. We reconstituted heterologous mouse (m) and human (h) TERT-TR complexes and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component.

A platinum supramolecular square as an effective G-quadruplex binder and telomerase inhibitor.

In this contribution, we report that a self-assembled platinum molecular square [Pt(en)(4,4'-dipyridyl)]4 can act as an efficient G-quadruplex binder and telomerase inhibitor. Molecular modeling studies show that the square arrangement of the four bipyridyl ligands, the highly electropositive nature of the overall complex, as well as hydrogen bonding interactions between the ethylenediamine ligands and phosphates of the DNA backbone all contribute to the observed strong binding affinity to the G-quadruplex. Through thermal denaturation studies with duplex and quadruplex FRET probes and enzymatic assays, we demonstrate that this platinum square strongly binds to G-quadruplexes and can act as an inhibitor of telomerase.

Platinum phenanthroimidazole complexes as G-quadruplex DNA selective binders.

Complexes that bind and stabilize G-quadruplex DNA structures are of significant interest due to their potential to inhibit telomerase and halt tumor cell proliferation. We here report the synthesis of the first Pt(II) G-quadruplex selective molecules, containing pi-extended phenanthroimidazole ligands. Binding studies of these complexes with duplex and quadruplex d(T(4)G(4)T(4))(4) DNA were performed.

Harnessing telomerase in cancer therapeutics.

Telomerase is an attractive target for anti-cancer therapeutics due to its requirement for cellular immortalization and expression in greater than 85% of human neoplasms. Though initially promising, strategies that inhibit telomerase with either small molecules or antisense oligonucleotides have a major limitation, namely the lag time required for telomere shortening before cellular effects are attained. As alternative approaches, immunotherapy and gene therapy have been tailored to exploit, rather than antagonize telomerase expression and/or activity.