Trends in microRNA detection

MicroRNAs (miRNAs) are short, ~22 nucleotide length RNAs that perform gene regulation. Recently, miRNA has been shown to be linked with the onset of cancer and other diseases based on miRNA expression levels. It is important, therefore, to understand miRNA function as it pertains to disease onset; however, in order to fully understand miRNA’s role in a disease, it is necessary to detect the expression levels of these small molecules. The most widely used miRNA detection method is Northern blotting, which is considered as the standard of miRNA detection methods. This method, however, is time-consuming and has low sensitivity. This has led to an increase in the amount of detection methods available. These detection methods are either solid phase, occurring on a solid support, or solution phase, occurring in solution. While the solid-phase methods are adaptable to high-throughput screening and possess higher sensitivity than Northern blotting, they lack the ability for in vivo use and are often time-consuming. The solution-phase methods are advantageous in that they can be performed in vivo, are very sensitive, and are rapid; however, they cannot be applied in high-throughput settings. Although there are multiple detection methods available, including microarray technology, luminescence-based assays, electrochemical assays, etc., there is still much work to be done regarding miRNA detection. The current gaps of miRNA detection include the ability to perform multiplex, sensitive detection of miRNA with single-nucleotide specificity along with the standardization of these new methods. Current miRNA detection methods, gaps in these methods, miRNA therapeutic options, and the future outlook of miRNA detection are presented here.     

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.     

PNIPAM chain collapse depends on the molecular weight and grafting density

This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacrylamide) (PNIPAM) above the lower critical solution temperature (LCST) of 32 degrees C depends on the chain grafting density and molecular weight. The polymer was grafted from the surface of a self-assembled monolayer containing the initiator (BrC(CH3)2COO(CH2)11S)2, using surface-initiated atom transfer radical polymerization. Varying the reaction time and monomer concentration controlled the molecular weight, and diluting the initiator in the monolayer altered the grafting density. Surface force measurements of the polymer films showed that the chain collapse above the LCST decreases with decreasing grafting density and molecular weight. At T > LCST, the advancing water contact angle increases sharply on PNIPAM films of high molecular weight and grafting density, but the change is less pronounced with films of low-molecular-weight chains at lower densities. Below the LCST, the force-distance profiles exhibit nonideal polymer behavior and suggest that the brush architecture comprises dilute outer chains and much denser chains adjacent to the surface.     

Carbenylative amination with N-tosylhydrazones

A Pd-catalyzed reaction of vinyl iodides and N-tosylhydrazones that assembles η(3)-allyl ligands through carbene insertion is demonstrated. Intramolecular trapping with nitrogen nucleophiles generates good yields of cinnamyl and pentadienyl amines like those found in alkaloid natural products. Carbenylative amination was the key reaction to complete the synthesis of the alkaloid caulophyllumine B. Migratory insertion was biased to provide allylamines with optical purity up to 64% ee, but in a lower yield.     

Catalytic enantioselective halolactonization of enynes and alkenes

New organocatalysts have been developed for the enantioselective halolactonization of (Z)-1,3-enynes and 1,1-disubstituted alkenes. In the case of 1,3-enynes, the carboxylate nucleophile and halogen electrophile were added to the conjugated π-system from the same face. Up to 99% ee was achieved for the 1,4-syn-bromolactonization of conjugated (Z)-1,3-enynes. Based on the results from the enyne halolactonization, a second generation of catalysts was designed for simple olefins. Up to 91% ee was observed for chlorolactonization of 1,1-disubstituted alkenes. The catalysts developed for the enantioselective halolactonization of both enynes and alkenes are composed of a cinchona alkaloid skeleton tethered to a urea group.     

Solution-phase detection of dual microRNA biomarkers in serum

A strategy for the simultaneous detection of multiple microRNA (miRNA) targets was developed utilizing fluorophore/quencher-labeled oligonucleotide probe sets. Two miRNA targets (miR-155 and miR-103), whose misregulation has afforded them status as putative biomarkers in certain types of cancer, were detected using our assay design. In the absence of target, the complementary fluorophore-probe and quencher-probe hybridize, resulting in a fluorescence resonance energy transfer-based quenching of the fluorescence signal. In the presence of unlabeled target, however, the antisense quencher-probe can hybridize with the target, resulting in increased fluorescence intensity as the quencher-probe is sequestered beyond the Förster radius of the fluorescent-probe. The assay design was tested in multiple matrices of buffer, cellular extract, and serum; and detection limits were found to be matrix-dependent, ranging from 0.34 to 8.89 pmol (3.4–59.3 nM) for miR-155 and 2.90–11.8 pmol (19.3–79.0 nM) for miR-103. Single, double, and triple nucleotide selectivity was also tested. Additionally, miR-155 concentrations were assessed in serum samples obtained directly from breast cancer patients without the need for RNA extraction. This assay is quantitative, possesses a low detection limit, can be applied in multiple complex matrices, and can obtain single-nucleotide selectivity. This method can be employed for the multiplex detection of solution-phase DNA or RNA targets and, more specifically, for the direct detection of serum miRNA biomarkers.     

Are Protein Force Fields Getting Better? A Systematic Benchmark on 524 Diverse NMR Measurements

Recent hardware and software advances have enabled simulation studies of protein systems on biophysically-relevant timescales, often revealing the need for improved force fields. Although early force field development was limited by the lack of direct comparisons between simulation and experiment, recent work from several labs has demonstrated direct calculation of NMR observables from protein simulations. Here we quantitatively evaluate recent molecular dynamics force fields against a suite of 524 chemical shift and J coupling ((3)JH(N)H(α), (3)JH(N)C(β), (3)JH(α)C', (3)JH(N)C', and (3)JH(α)N) measurements on dipeptides, tripeptides, tetra-alanine, and ubiquitin. Of the force fields examined (ff96, ff99, ff03, ff03*, ff03w, ff99sb*, ff99sb-ildn, ff99sb-ildn-phi, ff99sb-ildn-nmr, CHARMM27, OPLS-AA), two force fields (ff99sb-ildn-phi, ff99sb-ildn-nmr) combining recent side chain and backbone torsion modifications achieve high accuracy in our benchmark. For the two optimal force fields, the calculation error is comparable to the uncertainty in the experimental comparison. This observation suggests that extracting additional force field improvements from NMR data may require increased accuracy in J coupling and chemical shift prediction. To further investigate the limitations of current force fields, we also consider conformational populations of dipeptides, which were recently estimated using vibrational spectroscopy.     

Selectivity, directionality, and promiscuity in peptide processing from a Bacillus sp. Al Hakam cyclodehydratase

The thiazole/oxazole-modified microcins (TOMMs) represent a burgeoning class of ribosomal natural products decorated with thiazoles and (methyl)oxazoles originating from cysteines, serines, and threonines. The ribosomal nature of TOMMs allows for the generation of derivative products from mutations in the amino acid sequence of the precursor peptide, which ultimately manifest in differing structures and, sometimes, biological functions. Employing a TOMM system for the purpose of creating new structures and functions via combinatorial biosynthesis requires processing machinery that can tolerate highly variable substrates. In this study, TOMM enzymatic promiscuity was assessed using a currently uncharacterized cluster in Bacillus sp. Al Hakam. As determined by Fourier transform tandem mass spectrometry (FT-MS/MS), azole rings were formed in both a regio- and chemoselective fashion. Cognate and noncognate precursor peptides were modified in an overall C- to N-terminal directionality, which to date is unique among characterized ribosomal natural products. Studies focused on the inherent promiscuity of the biosynthetic machinery elucidated a modest bias for glycine at the preceding (-1) position and a remarkable flexibility in the following (+1) position, even allowing for the incorporation of charged amino acids and bisheterocyclization. Two unnatural substrates were utilized as the conclusive test of substrate flexibility, of which both were processed in a predictable fashion. A greater understanding of substrate processing and enzymatic tolerance toward unnatural substrates will prove beneficial when designing combinatorial libraries to screen for artificial TOMMs that exhibit desired activities.     

Total synthesis of oxidized welwitindolinones and (-)-N-methylwelwitindolinone C isonitrile

We report the total synthesis of (-)-N-methylwelwitindolinone C isonitrile, in addition to the total syntheses of the 3-hydroxylated welwitindolinones. Our routes to these elusive natural products feature the strategic use of a deuterium kinetic isotope effect to improve the efficiency of a late-stage nitrene insertion reaction. We also provide a computational prediction for the stereochemical configuration at C3 of the hydroxylated welwitindolinones, which was confirmed by experimental studies.