Advances in Quantitative FRET‐Based Methods for Studying Nucleic Acids

Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for “on/off” experiments. To use FRET to obtain quantitative distances and three‐dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single‐molecule levels.     

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids,Amido-Bridged Nucleic Acids,and Guanidine-Bridged Nucleic Acids

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (T_m) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same T_m values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower T_m values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.     

Circular Nucleic Acids: Discovery,Functions and Applications

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated.     

混合表面活性剂介质中微量核酸的光谱测定

健那绿（Janus Green B,JGB）与核酸形成离子性缔合物增溶于混合非离子性表面活性剂中,导致吸光度降低,降低程度与加入的核酸浓度成线性关系,在最佳反应条件下,小牛胸腺DNA（ctDNA）、鱼精子DNA（fsDNA）及酵母RNA（yRNA）的线性范围分别为0．0～4．2μg／mL,0．0～2．4μg／mL及0．0～1．9μg／mL,检测限分别为ctDNA：57ng／mL,fsDNA：96ng／mL及yRNA：42ng／mL,用双倒数直线方程求出各种核酸与JGB的结合常数,均为10^4数量级,本法已用于实际样品分析。     

转基因大豆的安全性与核酸检测技术的进展

转基因大豆通常指通过基因技术获得了抗草甘膦农药外源基因的大豆品种,相对于传统大豆,其核酸组成发生了一定的变化.总结了对于转基因大豆安全性的认识--关于转基因大豆的食品安全性,目前在世界范围内仍然存在部分争议.就转基因大豆和食品核酸检测技术的最新进展作了综述,指出聚合酶链式扩增反应(PCR)技术和改进的PCR技术仍然是主流检测技术,但是基因芯片等技术的发展为快速进行转基因大豆的核酸检测提供了更多的选择.     

Organomercury Nucleic Acids: Past,Present and Future

Synthetic efforts towards nucleosides, nucleotides, oligonucleotides and nucleic acids covalently mercurated at one or more of their base moieties are summarized, followed by a discussion of the proposed, realized and abandoned applications of this unique class of compounds. Special emphasis is given to fields in which active research is ongoing, notably the use of Hg^II-mediated base pairing to improve the hybridization properties of oligonucleotide probes. Finally, this minireview attempts to anticipate potential future applications of organomercury nucleic acids.     

Multivalent Interactions between an Aromatic Helical Foldamer and a DNA G‐Quadruplex in the Solid State

Quinoline‐based oligoamide foldamers have been identified as a potent class of ligands for G‐quadruplex DNA. Their helical structure is thought to target G‐quadruplex loops or grooves and not G‐tetrads. We report a co‐crystal structure of the antiparallel hairpin dimeric DNA G‐quadruplex (G₄T₄G₄)₂ with tetramer 1 —a helically folded oligo‐quinolinecarboxamide bearing cationic side chains—that is consistent with this hypothesis. Multivalent foldamer–DNA interactions that modify the packing of (G₄T₄G₄)₂ in the solid state are observed.     

Principles of Amino-Acid-Nucleotide Interactions Revealed by Binding Affinities between Homogeneous Oligopeptides and Single-Stranded DNA Molecules

We have determined the binding strengths between nucleotides of adenine, thymine, guanine and cytosine in homogeneous single stranded DNAs and homo-octapeptides consisting of 20 common amino acids. We use a bead-based fluorescence assay for these measurements in which octapeptides are immobilized on the bead surface and ssDNAs are in solutions. Comparative analyses of the distribution of the binding energies reveal unique binding strength patterns assignable to each DNA nucleotide and amino acid originating from the chemical structures. Pronounced favorable (such as Arg-G, etc.) and unfavorable (such as Ile-T, etc.) binding interactions can be identified in selected groups of amino acid and nucleotide pairs that could provide basis to elucidate energetics of amino-acid-nucleotide interactions. Such interaction selectivity, specificity and polymorphism establish the contributions from DNA backbone, DNA bases, as well as main chain and side chain of the amino acids.     

Triplex-Forming Peptide Nucleic Acids with Extended Backbones

Peptide nucleic acid (PNA) forms a triple helix with double-stranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (N−H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching ∼5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one −CH₂− group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional −CH₂− group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.     

Functional Nucleic Acids Under Unusual Conditions

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications.     

Palladium‐Mediated Labeling of Nucleic Acids

New applications of Pd‐catalyzed coupling reactions (Suzuki–Miyaura, Sonogashira, and Stille–Migita coupling) for post‐conjugation of nucleic acids have been developed recently. Breakthroughs in this area might now pave the way for the development of sophisticated DNA probes, which might be of great interest in chemical biology, nanotechnology, and bioanalysis, as well as in diagnostic domains.     

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H₂O₂ is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.     

Fluorobenzene Nucleobase Analogues for Triplex-Forming Peptide Nucleic Acids

2,4-Difluorotoluene is a nonpolar isostere of thymidine that has been used as a powerful mechanistic probe to study the role of hydrogen bonding in nucleic acid recognition and interactions with polymerases. In the present study, we evaluated five fluorinated benzenes as nucleobase analogues in peptide nucleic acids designed for triple helical recognition of double helical RNA. We found that analogues having para and ortho fluorine substitution patterns (as in 2,4-difluorotoluene) selectively stabilized Hoogsteen triplets with U−A base pairs. The results were consistent with attractive electrostatic interactions akin to non-canonical F to H−N and C−H to N hydrogen bonding. The fluorinated nucleobases were not able to stabilize Hoogsteen-like triplets with pyrimidines in either G−C or A−U base pairs. Our results illustrate the ability of fluorine to engage in non-canonical base pairing and provide insights into triple helical recognition of RNA.     

Current Challenges in Nucleic Acid Synthesis

Our understanding of the role of nucleic acids in regulating the process of life is growing rapidly. As researchers delve deeper into the cellular activity of molecules such as microRNA, small interfering RNA, and long non-coding RNA, the oligonucleotide chemist is confronted with the challenge of synthesizing the tools needed to continue this work. Furthermore, new applications are being developed that require increasingly complicated oligonucleotide constructs. In this review, we explore the current challenges confronting oligonucleotide synthesis, describe the chemistry and the enzymology being developed in response to those challenges, and discuss future challenges as-yet unmet.     

Flavin Conjugates for Delivery of Peptide Nucleic Acids

Oligonucleotides and their analogues, such as peptide nucleic acids (PNAs), can be used in chemical strategies to artificially control gene expression. Inefficient cellular uptake and inappropriate cellular localization still remain obstacles in biological applications, however, especially for PNAs. Here we demonstrate that conjugation of PNAs to flavin resulted in efficient internalization into cells through an endocytic pathway. The flavin–PNAs exhibited antisense activity in the sub‐micromolar range, in the context of a treatment facilitating endosomal escape. Increased endosomal release of flavin conjugates into the cytoplasm and/or nucleus was shown by chloroquine treatment and also—when the flavin–PNA was conjugated to rhodamine, a mild photosensitizer—upon light irradiation. In conclusion, an isoalloxazine moiety can be used as a carrier and attached to a cargo biomolecule, here a PNA, for internalization and functional cytoplasmic/nuclear delivery. Our findings could be useful for further design of PNAs and other oligonucleotide analogues as potent antisense agents.