Recognition Of Nucleic Acid Sequences Research Articles

PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.

Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).

6-Pyrazolylpurine as an Artificial Nucleobase for Metal-Mediated Base Pairing in DNA Duplexes.

The artificial nucleobase 6-pyrazol-1-yl-purine (6PP) has been investigated with respect to its usability in metal-mediated base pairing. As was shown by temperature-dependent UV spectroscopy, 6PP may form weakly stabilizing 6PP–Ag(I)–6PP homo base pairs. Interestingly, 6PP can be used to selectively recognize a complementary pyrimidine nucleobase. The addition of Ag(I) to a DNA duplex comprising a central 6PP:C mispair (C = cytosine) leads to a slight destabilization of the duplex. In contrast, a stabilizing 6PP–Ag(I)–T base pair is formed with a complementary thymine (T) residue. It is interesting to note that 6PP is capable of differentiating between the pyrimidine moieties despite the fact that it is not as sterically crowded as 6-(3,5-dimethylpyrazol-1-yl)purine, an artificial nucleobase that had previously been suggested for the recognition of nucleic acid sequences via the formation of a metal-mediated base pair. Hence, the additional methyl groups of 6-(3,5-dimethylpyrazol-1-yl)purine may not be required for the specific recognition of the complementary nucleobase.

On the feasibility of recognition of nucleic acid sequences by metal-ion-carrying oligonucleotides

Metal-ion-mediated base pairing has been extensively studied in the context of DNA nanotechnology or expansion of the genetic alphabet but only recently with the aim of recognition of natural nucleic acid sequences. This review article focuses on the attempts to utilize metal-ion-carrying oligonucleotides as high-affinity probes for nucleic acids but relevant studies carried out at the monomer level are also discussed. Special emphasis is given to challenges inherent to this approach, such as the limited recognition elements of the canonical nucleobases and the scarcity of suitable metal ions in the intracellular medium. Finally, potential ways to overcome these challenges are proposed.

The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.

ピロール‐イミダゾールポリアミドによるＤＮＡ塩基配列認識の進歩 ポスト・ゲノム時代の創薬

By the completion of the human genome project, many diseases including cancer, hereditary and viral diseases can be understood by the DNA sequence level. Control of the specific gene expression will provide ultimate gene therapy. Minor groove binding polyamides containing N-methylpyrrole and N-methylimidazole amino acids exhibit promising performance based on the recognition of nucleic acid sequences. Various types of sequence-specific DNA binding agents are developed and used for the regulation of gene expression. We synthesized novel type of polyamide-alkylator conjugates. These synthetic compounds alkylated predetermined DNA sequences selectively, and also some of them possessed selective potency for certain cancer cell lines. In this review, we will focus on recent progress of minor groove binding polyamides that play important roles in the rational recognition of nucleic acid sequences. One of the future directions of rational design of molecular medicine in the post genome era is proposed.

Rational recognition of nucleic acid sequences

Recent progress in synthetic and computational chemistry has made it possible to develop certain novel drug candidates. Drug candidates for genetic diseases, such as cancer, may also be designed on the basis of structural information obtained using X-ray analysis and NMR, as well as evidence from biological techniques applied to natural products–DNA (or RNA) complexes and conjugates. The resulting designed drug candidates exhibit promising performance based on the recognition on nucleic acid sequences.

The mode of Mg++binding to oligonucleotides. Inner sphere complexes as markers for recognition?

Large changes of UV absorbance and CD spectra as well as specific relaxation processes with time constrants around 50 mus are found for the association of Mg++ with A(pa)n. The Mg++ binding constants strongly increase with increasing n. The relaxation data demonstrate that a large fraction of Mg++ bound to short A(pA)n forms inner sphere complexes (ISC), with H2O molecules from the inner hydration sphere of Mg++ exchanged against some site (s) of the oligomer. This fraction decreases from about 85% for A(pA)4 to less than 10% for A(pA)17. A parallel decrease is observed in the relative change of CD spectrum upon Mg++ binding from 77.5% for A(pA)4 to 13.4% for (pA)17. The rate of ISC formation decreases with increasing n suggesting some (probably sterical) hindrance effect at high n. The data support the conclusion that Mg++ favours the formation of outer sphere complexes with linear polynucleotides and require a special chain folding for ISC. Measurements of Mg++ binding to C(pC)5, U(pU)5, I(pI)5 and d[A(pA)5] did not give evidence for the formation of ISC, indicating that both specific base and sugar residues are required for ISC. These results suggest the possibility that Mg++ISC ARE USED FOR SPECific recognition of nucleic acid sequences.