Actin exposure upon tissue injury is a targetable wound site-specific protein marker.

Background: Identification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.

A cell-free expression and purification process for rapid production of protein biologics.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics.

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome.

Background: Genetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively.

Peptide nucleic acids conjugated to short basic peptides show improved pharmacokinetics and antisense activity in adipose tissue.

A peptide nucleic acid (PNA) targeting a splice junction of the murine PTEN primary transcript was covalently conjugated to various basic peptides. When systemically administered to healthy mice, the conjugates displayed sequence-specific alteration of PTEN mRNA splicing as well as inhibition of full length PTEN protein expression. Correlating activity with drug concentration in various tissues indicated strong tissue-dependence, with highest levels of activity observed in adipose tissue.

Inhibitor-induced structural change in the HCV IRES domain IIa RNA.

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90 degrees helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication.

Evaluation of basic amphipathic peptides for cellular delivery of antisense peptide nucleic acids.

Cellular permeation peptides have been used successfully for the delivery of a variety of cargoes across cellular membranes, including large hydrophilic biomolecules such as proteins, oligonucleotides, or plasmid DNA. For the present work, a series of short amphipathic peptides was designed to elucidate the structural requirements for efficient and nontoxic delivery of peptide nucleic acids (PNAs). On the basis of an idealized alpha-helical structure, the helical parameters were modulated systematically to yield peptides within a certain range of hydrophobicity and amphipathicity.

Trisaccharide mimetics of the aminoglycoside antibiotic neomycin.

A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range.

Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability.

SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.

A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct.

Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid.

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages.

An efficient synthesis of gougerotin and related analogues using solid- and solution-phase methodology.

The first solid-phase synthesis of the natural product gougerotin has been accomplished. The synthetic route is versatile and allows for diversification at position C-4 of the heterocycle, C-6' of the sugar ring, and both residues of the peptidic moiety at N-4' in a parallel fashion. [structure: see text]

Positional effect of chemical modifications on short interference RNA activity in mammalian cells.

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones.

Rapid identification of emerging pathogens: coronavirus.

We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp.

Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

Disaccharide mimetics of the aminoglycoside antibiotic neamine.

A highly convergent approach has been employed for the facile synthesis of a library of 24 disaccharides that are alpha(1-3), beta(1-3), alpha(1-4), or beta(1-4) linked and contain 2-4 amino groups. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to determine dissociation constant (Kd) values for the binding of the disaccharides to a prototypical fragment of 16S ribosomal RNA. Several derivatives bound with affinities similar to that of neamine.

Multitarget affinity/specificity screening of natural products: finding and characterizing high-affinity ligands from complex mixtures by using high-performance mass spectrometry.

In this work we describe a high-throughput screening approach based on electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) that rapidly interrogates the noncovalent interaction between RNA-based drug targets and components derived from a bacterial natural product library. The screening process detects molecules present in the natural product library that bind to a synthetic RNA target that mimics the prokaryotic 16S rRNA A-site, while simultaneously measuring specificity for the synthetic A-site target using a control RNA target that lacks the critical structural element of the A-site construct. This screening approach known as multitarget affinity/specificity screening (MASS) demonstrated the expected binding of paromomycin from a fractionated natural product library derived from Streptomyces rimosus sp.

Discovery of RNA structural elements using evolutionary computation.

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures, or certain structural motifs within them, are thought to recur in multiple genes within a single organism or across the same gene in several organisms and provide a common regulatory mechanism. Search algorithms, such as RNAMotif, can be used to mine nucleotide sequence databases for these repeating motifs.

Converting solution macromolecular thermodynamic properties into gas-phase mass spectrometry observations.

Solution dissociation constants and changes in free energies associated with ligand binding to proteins have been measured in the gas phase using mass spectrometry.

SAR by MS: a ligand based technique for drug lead discovery against structured RNA targets.

A technique for lead discovery vs RNA targets utilizing mass spectrometry (MS) screening methods is described. The structure-activity relationships (SAR) derived from assaying weak binding motifs allows the pharmacophores discovered to be elaborated via "SAR by MS" to higher affinity ligands. Application of this strategy to a subdomain of the 23S rRNA afforded a new class of compounds with functional activity.

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell.