Journal of Nucleic Acids最新文献

Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA) as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.

Among the 518 protein kinases encoded by the human kinome, several of them act as oncoproteins in human cancers. Like other eukaryotic genes, oncogenes encoding protein kinases are frequently subjected to alternative splicing in coding as well as noncoding sequences. In the present paper, we will illustrate how alternative splicing can significantly impact on the physiological functions of oncogenic protein kinases, as demonstrated by mouse genetic model studies. This includes examples of membrane-bound tyrosine kinases receptors (FGFR2, Ret, TrkB, ErbB4, and VEGFR) as well as cytosolic protein kinases (B-Raf). We will further discuss how regular alternative splicing events of these kinases are in some instances implicated in oncogenic processes during tumor progression (FGFR, TrkB, ErbB2, Abl, and AuroraA). Finally, we will present typical examples of aberrant splicing responsible for the deregulation of oncogenic kinases activity in cancers (AuroraB, Jak2, Kit, Met, and Ron).

In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5' UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library.

We studied the effect of genetic susceptibility on hexavalent chromium induced dermal adversities. The health status of population was examined from the areas of Kanpur (India) having the elevated hexavalent chromium levels in groundwater. Blood samples were collected for DNA isolation to conduct polymorphic determination of genes, namely: NQO1 (C609T), hOGG1 (C1245G), GSTT1, and GSTM1 (deletion). Symptomatic exposed subjects (n = 38) were compared with asymptomatic exposed subjects (n = 108) along with asymptomatic controls (n = 148) from a non contaminated reference community. Exposed symptomatic group consisted of 36.8% subjects who were GSTM1 null genotyped as compared to asymptomatic where only 19.4% subjects were null. The exposed subjects with GSTM1 null genotype were more susceptible to dermal adversities in comparison with wild genotyped subjects (OR = 2.42; 95% CI = 1.071-5.451). Age, smoking, gender or duration of residence were not found to have any confounding effect towards this association. Association with other genes was not statistically significant, nonetheless, possible contribution by these genes cannot be ruled out. In conclusion, variation in the polymorphic status of GSTM1 gene may influence dermal outcomes among residents from Cr(VI) contaminated areas. Further studies are therefore, needed to examine these observations among different population groups.

Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets.

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression by modulating numerous target mRNAs expression at posttranscriptional level. Extensive studies have shown that miRNAs are critical in various important biological processes, including cell growth, proliferation, differentiation, development, and apoptosis. In terms of their importance, miRNA dysfunction has been associated with a broad range of diseases. Increased number of studies have shown that miRNAs can functionally interact with a wide spectrum of environmental factors (EFs) including drugs, industrial materials, virus and bacterial pathogens, cigarette smoking, alcohol, nutrition, sleep, exercise, stress, and radiation. More importantly, the interactions between miRNAs and EFs have been shown to play critical roles in determining abnormal phenotypes and diseases. In this paper, we propose an outline of the current knowledge about specific roles of miRNAs in their interactions with various EFs and analyze the literatures detailing miRNAs-EFs interactions in the context of various of diseases.

Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.

Folded RNA molecules have recently emerged as critical regulatory elements in biological pathways, serving not just as carriers of genetic information but also as key components in enzymatic assemblies. In particular, the transactivation response element (TAR) of the HIV genome regulates transcriptional elongation by interacting specifically with the Tat protein, initiating the recruitment of the elongation complex. Preventing this interaction from occurring in vivo halts HIV replication, thus making RNA-binding molecules an intriguing pharmaceutical target. Using α-amino acids as starting materials, we have designed and synthesized a new class of polyamino-amido oligomers, called PAAs, specifically for binding to folded RNA structures. The PAA monomers were readily incorporated into a 125-member combinatorial library of PAA trimers. In order to rapidly assess RNA binding, a quantum dot-based fluorescent screen was developed to visualize RNA binding on-resin. The binding affinities of hits were quantified using a terbium footprinting assay, allowing us to identify a ligand (SFF) with low micromolar affinity (k(d) = 14 μM) for TAR RNA. The work presented herein represents the development of a flexible scaffold that can be easily synthesized, screened, and subsequently modified to provide ligands specific for binding to folded RNAs.