High‐Performance Nucleic Acid Sensors for Liquid Biopsy Applications

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This “liquid biopsy” approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild‐type sequences. This review discusses high‐performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing‐ and amplification‐based methods to next‐generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis‐based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present.     

Enzymatic Synthesis of 7′,5′‐Bicyclo‐DNA Oligonucleotides

The selection of artificial genetic polymers with tailor‐made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo‐DNA triphosphate (i.e., 7′,5′‐bc‐TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7′,5′‐bc‐TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA‐dependent DNA synthesis and potentially also XNA‐dependent XNA synthesis. However, DNA‐dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7′,5′‐bc‐TTP.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

Valency‐Controlled Framework Nucleic Acid Signal Amplifiers

Weak ligand–receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)‐programmed strategy to develop valence‐controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA‐programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence‐controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor‐relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3–5 orders of magnitude and improved dynamic range.     

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5‐Pyrene‐Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers

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.     

稀土氨基酸配合物与核酸的相互作用*

很多抗癌金属药物是以核酸为靶标。阐明小分子与核酸之间的相互作用对筛选具有高效选择性和低毒副作用的抗癌药物有重要意义。近年来,开发新型的具有对核酸序列特异性识别能力的抗癌药物己成为本领域的研究热点。稀土离子具有良好的磁学、光学、电学特性和配位能力,使稀土配合物成为新型药物试剂。然而,稀土离子在中性条件下易水解的特性极大地阻碍了稀土配合物对核酸分子识别的研究。近年来在近生理条件下合成的一系列镧系氨基酸配合物具有结构稳定、溶解性好等优点,解决了镧系离子易水解的问题。本文总结了目前关于镧系氨基酸配合物与核酸的相互作用及其序列选择性等方面的研究进展。     

An Efficient and Modular Route to Sequence‐Defined Polymers Appended to DNA

Inspired by biological polymers, sequence‐controlled synthetic polymers are highly promising materials that integrate the robustness of synthetic systems with the information‐derived activity of biological counterparts. Polymer–biopolymer conjugates are often targeted to achieve this union; however, their synthesis remains challenging. We report a stepwise solid‐phase approach for the generation of completely monodisperse and sequence‐defined DNA–polymer conjugates using readily available reagents. These polymeric modifications to DNA display self‐assembly and encapsulation behavior—as evidenced by HPLC, dynamic light scattering, and fluorescence studies—which is highly dependent on sequence order. The method is general and has the potential to make DNA–polymer conjugates and sequence‐defined polymers widely available.     

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

A useful ²J(N?H) coupling‐based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA‐binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T‐cell intracellular antigen‐1 (TIA‐1) protein. The pK_a values of the His96 ionizable groups were substantially higher in the complexes with short U‐rich RNA and T‐rich DNA oligonucleotides than those of the isolated TIA‐1 RRM2. Herein, the methodology applied to determine changes in pK_a of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA‐binding proteins that shuttle among different cellular compartments.     

Enzymatic Synthesis of Vancomycin-Modified DNA

Many potent antibiotics fail to treat bacterial infections due to emergence of drug-resistant strains. This surge of antimicrobial resistance (AMR) calls in for the development of alternative strategies and methods for the development of drugs with restored bactericidal activities. In this context, we surmised that identifying aptamers using nucleotides connected to antibiotics will lead to chemically modified aptameric species capable of restoring the original binding activity of the drugs and hence produce active antibiotic species that could be used to combat AMR. Here, we report the synthesis of a modified nucleoside triphosphate equipped with a vancomycin moiety on the nucleobase. We demonstrate that this nucleotide analogue is suitable for polymerase-mediated synthesis of modified DNA and, importantly, highlight its compatibility with the SELEX methodology. These results pave the way for bacterial-SELEX for the identification of vancomycin-modified aptamers.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

One‐Step Nucleic Acid Purification and Noise‐Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow‐Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point‐of‐care diagnostics. Currently, nucleic acid (NA) purification remains time‐consuming and labor‐intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one‐step, liquid‐phase NA purification that is simpler and faster than conventional solid‐phase extraction. By further re‐concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non‐specific amplification caused by non‐optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non‐optimal PCR designs, which is 10‐ and 1000‐fold fewer than those of the standard bench‐top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point‐of‐care diagnosis.     

Synthesis and Base Pairing Properties of 1′,5′‐Anhydro‐L‐Hexitol Nucleic Acids (L‐HNA)

Oligonucleotides composed of 1′,5′‐anhydro‐arabino‐hexitol nucleosides belonging to the L series (L ‐HNA) were prepared and preliminarily studied as a novel potential base‐pairing system. Synthesis of enantiopure L ‐hexitol nucleotide monomers equipped with a 2′‐(N⁶‐benzoyladenin‐9‐yl) or a 2′‐(thymin‐1‐yl) moiety was carried out by a de novo approach based on a domino reaction as key step. The L oligonucleotide analogues were evaluated in duplex formation with natural complements as well as with unnatural sugar‐modified oligonucleotides. In many cases stable homo‐ and heterochiral associations were found. Besides T_m measurements, detection of heterochiral complexes was unambiguously confirmed by LC‐MS studies. Interestingly, circular dichroism measurements of the most stable duplexes suggested that L ‐HNA form left‐handed helices with both D and L oligonucleotides.     

锁核酸分子信标在分子识别与生物分析中的应用

分子信标是一种荧光探针,闭合时呈发夹结构。其5'末端修饰荧光基团,3'末端修饰猝灭基团。当目标存在时,环部与目标结合,发夹打开,发出荧光。锁核酸是一类双环状寡核苷酸衍生物,能够遵循碱基互补配对原则与核酸结合。锁核酸分子信标技术,结合了分子信标无需分离未结合探针而直接检测的优势和锁核酸亲合力强、热稳定性好、抗酶切以及体内无毒等特点,在核酸检测方面具有灵敏度高、特异性好的独特优势,近年来得到广泛关注。本文介绍了锁核酸修饰分子信标的结构、功能、设计要点,及其研究现状和一些重要进展,并讨论了目前锁核酸分子信标在分子识别及生物分析中的应用及存在的问题和发展前景。