An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

Chirality transmission in macromolecular domains.

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.

Improved Cancer Targeting by Multimerizing Aptamers on Nanoscaffolds.

Aptamers are short single-stranded oligonucleotides selected to bind with high affinity and specificity to a target. In contrast to antibodies, aptamers can be produced in large-scale in vitro systems without the need for any biological agents, making them highly attractive as targeting ligands for bioimaging and drug delivery. For in vivo applications it is often desirable to multimerize the aptamers in order to increase their binding strength and overall specificity. Additional functionalities, such as imaging and therapeutic agents, as well as pharmacokinetic modifiers, need to be attached in a stoichiometric fashion. Herein, we present a robust method for assembly of up to three aptamers and a fluorophore in a single well-defined nanostructure. The process is entirely modular and can be applied to any aptamer requiring only a single reactive "click handle." Multimerization of two aptamers, A9g and GL21.T, previously shown to target cancer cells, led to a strong increase in cell uptake. A similar effect was observed for the prostate-specific membrane antigen (PSMA)-targeting A9g aptamer in mice where multivalent aptamer binding led to increased tumor specificity. Altogether, this method provides a platform for multimerization of aptamers with advantages in terms of combinatorial screening capacity and multifunctional design of nanomedicine.

Self-Assembly of DNA-Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity.

DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Synthetic, Structural, and Anticancer Activity Evaluation Studies on Novel Pyrazolylnucleosides.

The synthesis of novel pyrazolylnucleosides 3a-e, 4a-e, 5a-e, and 6a-e are described. The structures of the regioisomers were elucidated by using extensive NMR studies. The pyrazolylnucleosides 5a-e and 6a-e were screened for anticancer activities on sixty human tumor cell lines. The compound 6e showed good activity against 39 cancer cell lines. In particular, it showed significant inhibition against the lung cancer cell line Hop-92 (GI50 9.3 µM) and breast cancer cell line HS 578T (GI50 3.0 µM).

Oligonucleotide Binding to Non-B-DNA in MYC.

MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major MYC promoters P1 and P2. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.

The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study.

Locked nucleic acid (LNA) oligonucleotides bind DNA target sequences forming Watson-Crick and Hoogsteen base pairs, and are therefore of interest for medical applications. To be biologically active, such an oligonucleotide has to efficiently bind the target sequence. Here we used molecular dynamics simulations and electrophoresis mobility shift assays to elucidate the relation between helical structure and affinity for LNA-containing oligonucleotides. In particular, we have studied how LNA substitutions in the polypyrimidine strand of a duplex (thus forming a hetero duplex, i.e. a duplex with a DNA polypurine strand and an LNA/DNA polypyrimidine strand) enhance triplex formation. Based on seven polypyrimidine single strand oligonucleotides, having LNAs in different positions and quantities, we show that alternating LNA with one or more non-modified DNA nucleotides pre-organizes the hetero duplex toward a triple-helical-like conformation. This in turn promotes triplex formation, while consecutive LNAs distort the duplex structure disfavoring triplex formation. The results support the hypothesis that a pre-organization in the hetero duplex structure enhances the binding of triplex forming oligonucleotides. Our findings may serve as a criterion in the design of new tools for efficient oligonucleotide hybridization.

MicroRNA therapeutics: design of single-stranded miR-216b mimics to target KRAS in pancreatic cancer cells.

Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.

Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides.

BACKGROUND: Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness. RESULTS: In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression. CONCLUSIONS: The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.

Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity.

BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design. METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone. RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent. CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

Synergy of Two Highly Specific Biomolecular Recognition Events: Aligning an AT-Hook Peptide in DNA Minor Grooves via Covalent Conjugation to 2'-Amino-LNA.

Two highly specific biomolecular recognition events, nucleic acid duplex hybridization and DNA-peptide recognition in the minor groove, were coalesced in a miniature ensemble for the first time by covalently attaching a natural AT-hook peptide motif to nucleic acid duplexes via a 2'-amino-LNA scaffold. A combination of molecular dynamics simulations and ultraviolet thermal denaturation studies revealed high sequence-specific affinity of the peptide-oligonucleotide conjugates (POCs) when binding to complementary DNA strands, leveraging the bioinformation encrypted in the minor groove of DNA duplexes. The significant cooperative DNA duplex stabilization may pave the way toward further development of POCs with enhanced affinity and selectivity toward target sequences carrying peptide-binding genetic islands.

An Albumin-Oligonucleotide Assembly for Potential Combinatorial Drug Delivery and Half-Life Extension Applications.

The long blood circulatory property of human serum albumin, due to engagement with the cellular recycling neonatal Fc receptor (FcRn), is an attractive drug half-life extension enabling technology. This work describes a novel site-specific albumin double-stranded (ds) DNA assembly approach, in which the 3' or 5' end maleimide-derivatized oligodeoxynucleotides are conjugated to albumin cysteine at position 34 (cys34) and annealed with complementary strands to allow single site-specific protein modification with functionalized ds oligodeoxynucleotides. Electrophoretic gel shift assays demonstrated successful annealing of complementary strands bearing Atto488, 6-carboxyfluorescein (6-FAM), or a factor IXa aptamer to the albumin-oligodeoxynucleotide conjugate. A fluorometric factor IXa activity assay showed retained aptamer inhibitory activity upon assembly with the albumin and completely blocked factor IXa at a concentration of 100 nM for 2 hr. The assembled construct exhibited stability in serum-containing buffer and FcRn engagement that could be increased using an albumin variant engineered for higher FcRn affinity. This work presents a novel albumin-oligodeoxynucleotide assembly technology platform that offers potential combinatorial drug delivery and half-life extension applications.

LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures.

The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3'-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3'-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

A hyaluronic acid-based hydrogel enabling CD44-mediated chondrocyte binding and gapmer oligonucleotide release for modulation of gene expression in osteoarthritis.

Hyaluronic acid (HA) is an attractive biomaterial for osteoarthritis (OA) treatment due to inherent functional and compatibility properties as an endogenous knee joint component. In this work, we describe a HA-based hydrogel with the dual functionality of increased CD44-dependent chondrocyte binding and controlled release of gapmer antisense oligonucleotides for unassisted cellular entry and subsequent gene silencing activity. A Schiff base-mediated gelation method was used to produce a panel of hydrogels varying in the aldehyde-modified HA (900kDa) to chitosan ratios (3:7, 5:5 and 7:3) for identifying designs displaying optimal engagement of OA patient-derived CD44-expressing chondrocytes. Correlation was found between cell binding and CD44 expression, with maximal binding exhibited at a HA/chitosan ratio of 7:3, that was 181% higher than CD44-negative MCF-7 cell control cells. Transfection agent-free uptake into OA chondrocytes of fluorescent 13-mer DNA oligonucleotides with a flanked locked nucleic acid (LNA) gapmer design, in contrast to naked siRNA, was demonstrated by confocal and flow cytometric analysis. A sustained and complete release over 5days was found with the 7:3 hydrogel, in contrast, the 5:5 and 3:7 hydrogel released 60% and 43% of loaded gapmers, respectively over the same period. A COX-2-specific gapmer designed with maximal chondrocyte gene silencing (~70% silencing efficiency at 500nM compared with a mismatch gapmer sequence) resulted in effective COX-2 silencing over 14days in hydrogels seeded with OA chondrocytes, with significant difference exhibited between day 3 and 10. This work introduces a novel HA-based CD44-mediated cellular binding and gapmer controlled release platform to modulate cellular gene expression.

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression.

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.

Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.