Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.

Cryptophane-A, comprised of two cyclotriguaiacylenes joined by three ethylene linkers, is a prototypal organic host molecule that binds reversibly to neutral small molecules via London forces. Of note are trifunctionalized, water-soluble cryptophane-A derivatives, which exhibit exceptional affinity for xenon in aqueous solution. In this paper, we report high-resolution X-ray structures of cryptophane-A and trifunctionalized derivatives in crown-crown and crown-saddle conformations, as well as in complexes with water, methanol, xenon or chloroform. Cryptophane internal volume varied by more than 20% across this series, which exemplifies 'induced fit' in a model host-guest system.

Peptide-modified cryptophane enables sensitive detection of protein analytes using hyperpolarized ¹²⁹Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing α_vβ₃ integrin receptor, which is overexpressed in many human cancers. Cryptophane was functionalized with cyclic RGDyK peptide and Alexa Fluor 488 dye, and cellular internalization was monitored by confocal laser scanning microscopy. Competitive blocking assays confirmed cryptophane endocytosis through an α_vβ₃ integrin receptor-mediated pathway. The peptide–cryptophane conjugate was determined to be nontoxic in normal human lung fibroblasts by MTT assay at the micromolar cryptophane concentrations typically used for hyperpolarized ¹²⁹Xe NMR biosensing experiments. Flow cytometry revealed 4-fold higher cellular internalization in cancer cells overexpressing the integrin receptor compared to normal cells. Nanomolar inhibitory concentrations (IC₅₀ = 20–30 nM) were measured for cryptophane biosensors against vitronectin binding to α_vβ₃ integrin and fibrinogen binding to α_IIbβ₃ integrin. Functionalization of the conjugate with two propionic acid groups improved water solubility for hyperpolarized ¹²⁹Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the ¹²⁹Xe-cryptophane–cyclic RGDyK biosensor. Introduction of α_IIbβ₃ integrin receptor in detergent solution generated a new “ bound” ¹²⁹Xe biosensor peak that was shifted 4 ppm downfield from the “free” ¹²⁹Xe biosensor.

Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb , a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the ‘caged’ state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6 , were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature ( T _m ) upon UV irradiation (Δ T _m = −29°C). The most thermally stable conjugate, C6 ( T _m = 84°C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6 , whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5 . However, no photomodulation of c-MYB protein levels was observed with S-C6 , perhaps due to the greater stability of this duplex.