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.     

Novel biomaterials derived from deoxyribozyme and NAPzyme

We report the potential of a small Ca²⁺-dependent deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA cleavage reaction is also detected using the deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyme) with its RNA cleavage function in the absence of divalent metal ions.     

Efficient Hydrolysis of RNA by a PNA - Diethylenetriamine Adduct

The introduction of a urea bond linking a protected diethylenetriamine (DETA) unit and the terminal amino group of a resin-bound peptide nucleic acid (PNA) decamer gave access to a PNA - DETA adduct (shown here), which hydrolyzed the target 25-mer RNA rapidly and sequence specifically.     

Toehold-Mediated Strand Displacement in a Triplex Forming Nucleic Acid Clamp for Reversible Regulation of Polymerase Activity and Protein Expression

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.     

Nucleic acid encoding to program self-assembly in chemical biology

This tutorial review serves as an introduction to the use of oligonucleotides and in particular peptide nucleic acids (PNAs) to encode function beyond heredity. Applications in chemical biology are reviewed starting with the use of nucleic acid tags to program self-assembled microarrays of small and macromolecules, followed by the use of nucleic acid templated reactions for the purpose of DNA or RNA sensing and finally, the use of nucleic acid templates to display ligands.     

8-Vinylguanine nucleo amino acid: a fluorescent PNA building block

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.     

Nucleobase‐Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In Vitro and in Cells

Compounds that bind specifically to double‐stranded regions of RNA have potential as regulators of structure‐based RNA function; however, sequence‐selective recognition of double‐stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA–RNA triplexes. Herein, we demonstrate that a 9‐mer PNA forms a sequence‐specific PNA–RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5′ untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex‐forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double‐stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein–RNA interactions.     

Pyrrolidinyl peptide nucleic acid homologues: effect of ring size on hybridization properties

The effect of ring size of four- to six-membered cyclic β-amino acid on the hybridization properties of pyrrolidinyl peptide nucleic acid with an alternating α/β peptide backbone is reported. The cyclobutane derivatives (acbcPNA) show the highest T(m) and excellent specificity with cDNA and RNA.     

Incorporation of Pseudo‐complementary Bases 2,6‐Diaminopurine and 2‐Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid‐based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6‐diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA‐RNA duplexes. However, D incorporation into short self‐complementary regions of SNA promoted self‐dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2‐thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self‐hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA‐based probes and nucleic acid drugs.     

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.     

Cellular delivery of peptide nucleic acids and inhibition of human telomerase.

BACKGROUND: Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function. Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization. RESULTS: We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly. Relative potencies of inhibition delineate putative structural domains. We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs. CONCLUSIONS: PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents. The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes. Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.     

PNA探针与DNA探针的系统比较

肽核酸（Peptide Nucleic Acid,PNA）是近十几年发展起来的以中性酰胺键为骨架的脱氧核糖核酸（Deoxyribonucleic Acid,DNA）类似物,其结构介于多肽和DNA之间。由于PNA能够与DNA和RNA特异性地结合,可以制备PNA探针。与DNA探针相比,其杂交的稳定性和特异性增加且能在低盐浓度下进行杂交。本文从DNA和PNA的分子结构和性质、DNA探针和PNA探针的设计制备、杂交亲和性、杂交动力学以及在生物传感器上的应用等方面进行了系统比较。     

Discovering protein-binding RNA motifs with a generative model of RNA sequences

Recent advances in high-throughput experimental technologies have generated a huge amount of data on interactions between proteins and nucleic acids. Motivated by the big experimental data, several computational methods have been developed either to predict binding sites in a sequence or to determine if an interaction exists between protein and nucleic acid sequences. However, most of the methods cannot be used to discover new nucleic acid sequences that bind to a target protein because they are classifiers rather than generators. In this paper we propose a generative model for constructing protein-binding RNA sequences and motifs using a long short-term memory (LSTM) neural network. Testing the model for several target proteins showed that RNA sequences generated by the model have high binding affinity and specificity for their target proteins and that the protein-binding motifs derived from the generated RNA sequences are comparable to the motifs from experimentally validated protein-binding RNA sequences. The results are promising and we believe this approach will help design more efficient in vitro or in vivo experiments by suggesting potential RNA aptamers for a target protein.     

Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA

Guanidine-based peptide nucleic acid (GPNA) with a d-backbone configuration and alternate spacing binds sequence-specifically to RNA and is readily taken up by both human somatic and embryonic stem (ES) cells.     

肽核酸骨架分子键联卟啉的合成及结构表征

以5,10,15-三苯基-20-(4-羧基苯基)卟啉和5,10,15-三苯基-20-(4-羟基苯基)卟啉为原料, 分别与N-(Boc-氨乙基)甘氨酸乙酯(3)及其衍生物4作用, 得到了两种肽核酸骨架分子键联卟啉化合物6和8. 中间体和目标化合物均由紫外-可见光谱、红外光谱、核磁共振光谱、质谱及元素分析所确证. 目标化合物的荧光光谱测试结果表明, 肽核酸单元分子的链接对卟啉分子的荧光波长和强度影响不大.     

金属配合物-寡聚核苷酸定位断裂剂研究进展

核酸切割试剂与寡聚核苷酸(ODN)偶联制得的人工核酸酶能在特定位点断裂DNA或RNA,为人工核酸酶的分子设计提供了一种新方法.本文综述了金属配合物-ODN识别切割试剂的偶联方式及其与靶分子的作用机制,并指出了今后的研究方向.     

Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps

A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of microRNAs (miRNAs). After peptide nucleic acid (PNA) capture probes were immobilized in nanogaps of a pair of interdigitated microelectrodes and hybridization was performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline (PAn) nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the PAn nanowires onto the hybridized target miRNA. The conductance of the deposited PAn nanowires correlates directly to the amount of the hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5.0 fM. The biosensor array is applied to the direct detection of miRNA in total RNA extracted from cancer cell lines.     

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.     

Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level

We report the specific detection of a few hundred molecules of genetic material using a fluorescent polythiophene biosensor. Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target and can be done in less than 1 h, simply and affordably, and without any chemical reaction. This simple system is versatile enough to detect nucleic acids of various lengths, including a segment from the RNA genome of the Influenza virus.