Patrick J. Hrdlicka

Pyrene-functionalized oligonucleotides and locked nucleic acids (LNAs): tools for fundamental research, diagnostics, and nanotechnology.

Pyrene-functionalized oligonucleotides (PFOs) are increasingly explored as tools in fundamental research, diagnostics and nanotechnology. Their popularity is linked to the ability of pyrenes to function as polarity-sensitive and quenchable fluorophores, excimer-generating units, aromatic stacking moieties and nucleic acid duplex intercalators. These characteristics have enabled development of PFOs for detection of complementary DNA/RNA targets, discrimination of single nucleotide polymorphisms (SNPs), and generation of π-arrays on nucleic acid scaffolds. This critical review will highlight the physical properties and applications of PFOs that are likely to provide high degree of positional control of the chromophore in nucleic acid complexes. Particular emphasis will be placed on pyrene-functionalized Locked Nucleic Acids (LNAs) since these materials display interesting properties such as fluorescence quantum yields approaching unity and recognition of mixed-sequence double stranded DNA (144 references).

2′-N-(Pyren-1-yl)acetyl-2′-Amino-α-L-LNA: Synthesis and Detection of Single Nucleotide Mismatches in DNA and RNA Targets

Single nucleotide polymorphisms (SNPs) are the most prevalent type of genetic mutation in the human genome (>1.4 million SNPs), and are commonly associated with diseases and individual variations in response to therapeutics. Several SNP detection methods have been developed but there is a continued need to develop simple, rapid, and sensitive detection methods. The use of homogenous detection assays is an appealing approach as they exhibit fast hybridization kinetics, absence of time-consuming washing, separation, or purification steps, and offer the promise of in vivo detection. Dual-labeled oligonucleotide probes such as Magi, TaqMan or Invader probes and molecular beacons are prominent examples of homogenous assays for detection of SNPs. These probes rely on hybridization-induced distance changes between two optical units for detection of nucleic acid targets. Alternative, and intuitively less complicated probes for detections of SNPs include oligonucleotide (ON) probes modified with a single kind of fluorophore, such as phenanthridinium, fluorene, fluorescein, thiazole orange, fluorescent nucleobases, or pyrene, whose optical properties change according to the microenvironment of the fluorophore. The high thermal affinities of locked nucleic acid (LNA), 2’-amino-LNA, and a-L-LNA toward complementary DNA and RNA sequences are well established and have stimulated the development of 2’-amino-LNA and 2’-amino-a-L-LNA building blocks. N2’-Functionalized derivatives of these building blocks exhibit interesting properties for nucleic acid-based diagnostics and therapeutics as a consequence of the precise positioning of functional entities in nucleic acid duplexes. Whereas N2’-functionalized derivatives of 2’-amino-LNA position functional groups in the minor groove of duplexes, preliminary studies suggest that the 2-oxa-5-azabicyclo ACHTUNGTRENNUNG[2.2.1]heptane skeleton of 2’-amino-a-L-LNA positions the N2’-linked aromatic moieties in duplex cores. Here, we have used this property to develop 2’-N-(pyren-1-yl)acetyl-2’-amino-a-L-LNA probes that allow detection of DNA or RNA targets with single mismatched nucleotides. (We define 2’-N-(pyren-1-yl)acetyl-2’amino-a-L-LNA as an oligonucleotide that contains one or more 2’-N-(pyren-1-yl)acetyl-2’-amino-2’-deoxy-2’-N,4’-C-methylene-a-l-ribofuranosyl monomer(s).) Synthesis of target phosphoramidite 3 was achieved in two steps from known 5’-O-dimethoxytritylated-2’-amino-a-L-LNA nucleoside 1 (Scheme 1). Chemoselective N-acylation of nucleoside 1 mediated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) yielded nucleoside 2 as a rotameric mixture (~1:2.5 from H NMR) in excellent yield. Disappearance of H NMR signals from the exchangeable O3’ proton upon addition of D2O confirmed N2’ functionalization of compound 2. Subsequent O3’ phosphitylation under standard ACHTUNGTRENNUNGconditions afforded phosphoramidite 3 as a suitable building block for oligodeoxyribonucleotide synthesis, which was performed on a 0.2 mmol scale by using an automated DNA syn-

Sensitive SNP Dual-Probe Assays Based on Pyrene-Functionalized 2′-Amino-LNA: Lessons To Be Learned†

A homogenous fluorescence dual‐probe assay containing 2′‐N‐(pyren‐1‐ylmethyl)‐2′‐amino‐LNA (locked nucleic acid) building blocks has been developed for effective mismatch‐sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2′‐amino‐LNA through a short linker, are positioned at the 3′ and 5′ ends of a dual‐probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal‐denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch‐induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual‐probe assay constitutes a simple and sensitive system for the detection of single‐nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual‐probe constructs.

Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA’s oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.

Gold-peptide nanoconjugate cellular uptake is modulated by serum proteins

Gold nanoparticles (Au NPs, 20 nm) were conjugated with two different cysteine-terminated peptides. Radio-ligand binding studies were conducted to characterize Au NP-peptide binding, suggesting both covalent and noncovalent interactions. The interactions of serum proteins with Au NP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugates (15 minutes). The cellular uptake of free peptides and nanoconjugates into mouse myogenic (Sol8) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence signals compared to those in the presence of serum (P < 0.05), suggesting that serum proteins inhibit Au NP-mediated peptide delivery. The cellular uptake of nanoconjugates was also confirmed using transmission electron microscopy. These data suggest that Au NP-peptide nanoconjugates are a useful platform for intracellular delivery of therapeutics. However, a deeper understanding of the mechanisms regulating their uptake and intracellular trafficking is needed.

C5‐Functionalized LNA: Unparalleled Hybridization Properties and Enzymatic Stability

Antisense oligonucleotides (ONs) are widely explored as fundamental research tools and therapeutic agents against diseases of genetic origin due to their ability to modulate gene expression by interfering with target RNA. Introduction of chemically modified nucleotides into antisense ONs is crucial to increase binding affinity toward RNA targets, improve discrimination of mismatched RNA to avoid off-target effects, and enhance stability against nucleases to slow down degradation. The use of conformationally restricted nucleotides and locked nucleic acids (LNAs, Scheme 1) in particular, has to some extent addressed these challenges. Antisense LNAs are accordingly evaluated in several clinical trials. Substantial efforts have been invested to develop LNA analogues with even more desirable biophysical properties and reduced hepatotoxicity. These studies have primarily focused on modification of the oxymethylene bridge spanning the C2’and C4’-positions and/or introduction of minor-groove-oriented substituents into the bridge. Improved enzymatic stability, e, f, j] altered biodistribution, or reduced hepatotoxicity has been reported for some of the analogues, but improvements in hybridization properties relative to LNA were generally not observed. Results from comparative in vivo antisense studies must be awaited to assess if the significantly increased synthetic complexity of these conformationally restricted nucleotides is justified. C5-functionalized pyrimidine DNA building blocks have attracted considerable attention due to their ability to accommodate functional entities in the major groove of nucleic acid duplexes and straightforward synthesis. Small C5-entities are generally well tolerated in duplexes and result in small increases in thermal affinity toward DNA/RNA complements. f] In light of this, we hypothesized that C5-alkynyl-functionalized LNA monomers would synergistically integrate beneficial Scheme 1. Synthetic outline of phosphoramidites 5 W–5 Z. CAN = ceric ammonium nitrate, DMTr = 4,4’-dimethoxytrityl, TBAF = tetrabutylammonium fluoride, PCl = 2-cyanoethyl N, N’diisopropylchlorophosphoramidite.

Optimized DNA targeting using N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA

Incorporation of N,N-bis(2-pyridylmethyl)-β-alanyl 2′-amino-LNA (bipyridyl-functionalized 2′-amino locked nucleic acid) monomers into DNA strands enables high-affinity targeting of complementary DNA with excellent Watson–Crick selectivity in the presence of divalent metal ions. Positioning of bipyridyl-functionalized 2′-amino-LNA monomers in two complementary DNA strands in a “3′-end zipper” constitution allows modulation of duplex stability, i.e., a strong stabilizing effect with one equivalent of divalent metal ion per bipyridyl pair, or a strong destabilizing effect with an excess of divalent metal ions.

Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases.

Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger

Biotransformation of the sesquiterpenoid trans‐nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12‐hydroxy‐ trans‐nerolidol, a precursor in the synthesis of the industrially interesting flavor α‐sinensal. We characterized biotransformations of cis‐nerolidol, trans‐nerolidol, and a commercially available cis/ trans‐nerolidol mixture in repeated batch cultures of A. niger grown in computer‐controlled bioreactors. On‐line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (±)‐ cis/ trans‐nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12‐hydroxy‐ trans‐nerolidol (9%) was obtained by addition of a (±)‐ cis/trans‐nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.

Invaders: Recognition of Double‐Stranded DNA by Using Duplexes Modified with Interstrand Zippers of 2′‐O‐(Pyren‐1‐yl)methyl‐ribonucleotides

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.