No abstract

Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA, developed more than a decade ago in which the naturally occurring sugar phosphate backbone has been replaced by the N-(2-aminoethyl) glycine units. Unlike DNA or RNA in the unhybridized state (single strand) which can adopt a helical structure through base-stacking, although highly flexible, PNA does not have a well-defined conformational folding in solution. Herein, we show that a simple backbone modification at the gamma-position of the N-(2-aminoethyl) glycine unit can transform a randomly folded PNA into a helical structure. Spectroscopic studies showed that helical induction occurs in the C- to N-terminal direction and is sterically driven. This finding has important implication not only on the future design of nucleic acid mimics but also on the design of novel materials, where molecular organization and efficient electronic coupling are desired.

Photoacoustic imaging (PA) is continuing to be applied for physiological imaging and more recently for molecular imaging of living subjects. Owing to its high spatial resolution in deep tissues, PA imaging holds great potential for biomedical applications and molecular diagnostics. There is however a lack of probes for targeted PA imaging, especially in the area of enzyme-activatable probes. Here we introduce a molecular probe, which upon proteolytic processing is retained at the site of enzyme activity and provides PA contrast. The probe oligomerizes via a condensation reaction and accumulates in cells and tumors that express the protease. We demonstrate that this probe reports furin and furin-like activity in cells and tumor models by generating a significantly higher photoacoustic signal relative to furin-deficient and non-target controls. This probe could report enzyme activity in living subjects at depths significantly greater than fluorescence imaging probes and has potential for molecular imaging in deep tumors.

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycininduced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.optical imaging | reporter gene P rotein-protein interactions (PPIs) are a prerequisite to most cellular processes, and their pharmacologic control offers promising avenues for therapeutic intervention in a variety of diseases (1). Developing techniques to identify and analyze these transient protein-protein associations is, therefore, of high importance. Several methods are available to investigate PPIs in vitro and in cell culture (2, 3). However, ex vivo analysis of interacting proteins neglects the intricate effects of physiologic and pathophysiologic stimuli that occur in the native microenvironment within living organisms. Similarly, in vitro drug validation ignores the pharmacokinetic properties of prospective pharmacophores. To investigate PPIs in their actual biological context, imaging technologies that enable direct translation of cell culture assays to deep tissues of living subjects are required. Current approaches for monitoring PPIs rely on the two-hybrid system (4), FRET (5), split reporter protein complementation and reconstitution (6-8), and bioluminescence resonance energy transfer (BRET) (9-11). Because most of these PPI detection schemes are optical approaches, signal attenuation by tissues constitutes the primary impediment for studying PPIs in animal models, and it results in low sensitivity for these assays, especially in the context of deep-tissue tumor models.Bioluminescence-based methods hold particular promise for imaging of PPIs in small living subjects (12, 13). Although both BRET and FRET detection schemes rely on the Förster resonance energy transfer mechanism (14), BRET systems provide enhanced sens...

Purpose: To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma.Experimental Design: We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method.Results: Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent.Conclusions: With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.

A selenium analogue of amino‐D‐luciferin, aminoseleno‐D‐luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red‐shifted bioluminescence emission maximum at 600 nm (see scheme) and is suitable for bioluminescence imaging studies in living subjects.

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.

Furin, a proprotein convertases family endoprotease, processes numerous physiological substrates and is overexpressed in cancer and inflammatory conditions. Non-invasive imaging of furin activity will offer a valuable tool to probe the furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate D-aminoluciferin. In the presence of luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking experiments with a furin inhibitor and control experiments with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After the furin activation, a 30-fold increase in the bioluminescent emission was observed in vitro and on average 7-8 fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and discovery of new drugs for furin-targeted cancer therapy.