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.     

Enzymatic Incorporation of Modified Purine Nucleotides in DNA

A series of nucleotide analogues, with a hypoxanthine base moiety (8‐aminohypoxanthine, 1‐methyl‐8‐aminohypoxanthine, and 8‐oxohypoxanthine), together with 5‐methylisocytosine were tested as potential pairing partners of N⁸‐glycosylated nucleotides with an 8‐azaguanine or 8‐aza‐9‐deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5‐methylisocytosine nucleotide followed by the 1‐methyl‐8‐aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8‐azaguanine versus 8‐aza‐9‐deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8‐aza‐9‐deazaguanine represents a self‐complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed).     

Stabilisation of RNA bulges by oligonucleotide complements containing an adenosine analogue

Incorporation of 2'-deoxy-2'-beta-(1-naphthylmethyl)tubercidin into an oligodeoxyribonucleotide mostly has little or a slightly negative effect on the T(m) values of complexes with DNA complements. With the same naphthylmethyl-substituted nucleoside at the 3'-end of a 2'-O-methyloligoribonucleotide, however, a stabilisation of 1-2 degrees C in the corresponding complexes with both DNA and RNA is observed. When the target sequence is an RNA fragment forming a two- or three-nucleotide bulge, complexes with (naphthylmethyl)tubercidin-modified oligodeoxyribonucleotides, as well as with the corresponding 2'-O-methyloligoribonucleotides, give stabilisations of 1-2 degrees C for the three-nucleotide bulge and of almost 4 degrees C for the two-nucleotide bulge. This stabilisation is specific to RNA, since the corresponding complexes with the DNA fragments do not display this effect. Thus, the (naphthylmethyl)tubercidin-containing oligonucleotides are the first reported oligonucleotide modifications that specifically stabilise bulged RNA.     

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.     

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.     

Parallel DNA Synthesis on Poly(ethylene terephthalate)

The fabrication of DNA arrays directly on aminolyzed sheets of poly(ethylene terephthalate) (PET) is described. Array surfaces typically employ bifunctional linkers or layers of covalently attached polymers to provide substrate hydroxy groups as synthesis attachment points. An amine treatment is used here to expose hydroxy groups on films of PET. These hydroxy groups can then be used to couple phosphoramidites and initiate the array synthesis without further functionalization steps. Arrays fabricated on these substrates with a maskless array synthesizer are tolerant of the high number of chemical exposure steps required to synthesize relatively long oligonucleotides. The results might be of the greatest use to the synthetic biology community, for whom a flexible and robust substrate could enable new strategies to enhance the throughput of oligonucleotide synthesis.     

Bleomycin Can Cleave an Oncogenic Noncoding RNA

Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Herein, an in‐depth analysis of the RNA cleavage preferences of bleomycin A5 is presented. Bleomycin A5 prefers to cleave RNAs with stretches of AU base pairs. Based on these preferences and bioinformatic analysis, the microRNA‐10b hairpin precursor was identified as a potential substrate for bleomycin A5. Both in vitro and cellular experiments demonstrated cleavage. Importantly, chemical cleavage by bleomycin A5 in the microRNA‐10b hairpin precursors occurred near the Drosha and Dicer enzymatic processing sites and led to destruction of the microRNA. Evidently, oncogenic noncoding RNAs can be considered targets of cancer medicines and might elicit their pharmacological effects by targeting noncoding RNA.     

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis,Hybridization Studies,Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5‐(pyren‐1‐yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene‐modified nucleobase component were found to destabilize an i‐motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene‐modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene‐modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady‐state fluorescence emission studies of oligonucleotides containing pyrene‐modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene‐modified UNA monomers was observed after formation of i‐motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene‐modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.     

Structural Impact of Single Ribonucleotide Residues in DNA

Single ribonucleotide intrusions represent the most common nonstandard nucleotide type found incorporated in genomic DNA, yet little is known of their structural impact. This lesion incurs genomic instability in addition to affecting the physical properties of the DNA. To probe for structural and dynamic effects of single ribonucleotides in various sequence contexts—AxC, CxG, and GxC, where x=rG or dG—we report the structures of three single‐ribonucleotide‐containing DNA duplexes and the corresponding DNA controls. The lesion subtly and locally perturbs the structure asymmetrically on the 3′ side of the lesion in both the riboguanosine‐containing and the complementary strand of the duplex. The perturbations are mainly restricted to the sugar and phosphodiester backbone. The ribose and 3′‐downstream deoxyribose units are predominately in N‐type conformation; backbone torsion angles ? and/or ζ of the ribonucleotide or upstream deoxyribonucleotide are affected. Depending on the flanking sequences, the C2′?OH group forms hydrogen bonds with the backbone, 3′‐neighboring base, and/or sugar. Interestingly, even in similar purine‐rG‐pyrimidine environments (A‐rG‐C and G‐rG‐C), a riboguanosine unit affects DNA in a distinct manner and manifests different hydrogen bonds, which makes generalizations difficult.