ABSTRACT
Oligodeoxyribonucleotides modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non-stringent conditions, relative to the corresponding LNA-free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore-labeled probes for SNP-discrimination studies.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Oligonucleotides/chemistry , Polymorphism, Single Nucleotide , Fluorescence , Oligonucleotides/chemical synthesisABSTRACT
LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides. We hypothesized that the LNA monomers would alter the sugar rings of the modified DNA monomers toward more RNA-like North-type conformations for maximal DNA/RNA affinity and specificity. Indeed, the incorporation of LNA monomers almost invariably results in increased target affinity and specificity relative to the corresponding LNA-free ONs, but the magnitude of the stabilization varies greatly. Introduction of LNA nucleotides as direct neighbors into C5-pyrene-functionalized pyrimidine DNA monomers yields oligonucleotide probes with more desirable photophysical properties as compared to the corresponding LNA-free probes, including more intense fluorescence emission upon target binding and improved discrimination of single nucleotide polymorphisms (SNPs). These hybrid oligonucleotides are therefore promising probes for diagnostic applications.
Subject(s)
DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Oligonucleotides/chemistry , Alkynes/chemistry , Base Sequence , DNA/genetics , Nucleic Acid Hybridization , RNA/chemistry , Spectrometry, FluorescenceABSTRACT
Self-assembled monolayers (SAMs) of thiols of L-cysteine, 6-mercaptohexanol, 4-mercaptobenzoic acid, DL-thioctic acid and 11-(1-pyrenyl)-1-undecathiol, which have been selected for their propensity to interact with vaporized explosives, have been attached from solution onto gold decorated ZnO-coated nanosprings. X-ray and ultraviolet photoelectron spectroscopies (XPS and UPS) have been used to investigate the surface electronic structure of the SAMs coated nanosprings. On the basis of XPS analysis, it has been determined that the packing densities of L-cysteine, 6-mercaptohexanol, 4-mercaptobenzoic acid, DL-thioctic acid and 11-(1-pyrenyl)-1-undecathiol on gold (zinc oxide) are 5.42 × 10(14) (2.83 × 10(14)), 3.26 × 10(14) (2.54 × 10(14)), 9.50 × 10(13), 2.55 × 10(14) (1.12 × 10(14)), and 5.23 × 10(13) molecules/cm(2), respectively. A single S 2p core level doublet is observed for 4-mercaptobenzoic acid and 11-(1-pyrenyl)-1-undecathiol, which is assigned to the S-Au bond. The S 2p core level for L-cysteine, 6-mercaptohexanol, and DL-thioctic acid consist of two doublets, where one is S-Au bond and the other is the S-Zn bond. Analysis of the C/S ratios agrees well with the stoichiometry of the respective thiols. UPS analysis shows that the hybridization of S 3p states and Au d-bands produces antibonding and bonding states, above and below the Au d-bands, which is characteristic of molecular chemisorption on Au nanoparticles. Gas sensors were constructed with thiolated nanosprings and their responsiveness to ammonium nitrate at 100-150 °C was tested. Nanosprings sensors functionalized with 4-mercaptobenzoic acid and 6-mercaptohexanol showed the strongest responses by a factor of 4 to 5 relative to the less responsive thiols. The response to ammonium nitrate can be correlated to the packing density and ordering of the SAMs.
ABSTRACT
Three different C5-carbohydrate-functionalized LNA uridine phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides. C5-Carbohydrate-functionalized LNA display higher affinity toward complementary DNA/RNA targets (ΔTm/modification up to +11.0 °C), more efficient discrimination of mismatched targets, and superior resistance against 3'-exonucleases compared to conventional LNA. These properties render C5-carbohydrate-functionalized LNAs as promising modifications in antisense technology and other nucleic acid targeting applications.
Subject(s)
Carbohydrates/chemistry , DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Oligonucleotides/chemistry , Organophosphorus Compounds/chemical synthesis , Uridine/analogs & derivatives , Molecular Structure , Nucleic Acid Conformation , Oligonucleotides, Antisense/chemistry , Organophosphorus Compounds/chemistry , Uridine/chemical synthesis , Uridine/chemistryABSTRACT
Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.
Subject(s)
Adenosine/chemistry , Cytidine/chemistry , DNA/chemistry , Nucleic Acid Hybridization/methods , Nucleic Acids/chemical synthesis , Oligonucleotides/chemical synthesis , Fluorescence , Nucleic Acid Conformation , Nucleic Acids/chemistry , Oligonucleotides/chemistryABSTRACT
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.
Subject(s)
Nucleic Acids/chemistry , Oligodeoxyribonucleotides/chemistry , Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/chemical synthesis , RNA/chemistry , Uridine/analogs & derivatives , Molecular Conformation , Nucleic Acid Conformation , Uridine/chemical synthesis , Uridine/chemistryABSTRACT
Oligonucleotides modified with consecutive incorporations of 5-(1-aryl-1,2,3-triazol-4-yl)-2'-deoxyuridine monomers display strong thermal affinity and binding specificity toward RNA targets, due to formation of chromophore arrays in the major groove.
