A microtube screening approach affords simple and convenient assessment of the selective adsorption of metal impurities by a variety of different process adsorbents. This approach is helpful in identifying rapid solutions to metal impurity problems in pharmaceutical process research. Several examples illustrating the utility of the approach are presented.

The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.

Bioconjugates have been used to deliver therapeutic oligonucleotides to their pharmacological targets in diseased cells. Molecular-scale conjugates can be prepared by directly linking targeting ligands with oligonucleotides and the resultant conjugates can selectively bind to cell surface receptors in target cells in diseased tissues. Besides targeted delivery, additional functionality can be incorporated in the conjugates by utilization of carrier molecules, and these larger conjugates are called carrier-associated conjugates. Both molecular and carrier-associated conjugates have achieved initial successes in clinical trials for treating liver diseases; therefore, currently the greater challenge is to deliver oligonucleotides to extrahepatic tissues such as tumors. This review will provide an update on the application of oligonucleotide conjugates for targeted delivery during the last decade. By identifying key elements for successful delivery, it is suggested that oligonucleotide conjugates with intermediate size, cell targeting ability, and endosomal release functionality are superior systems to advance oligonucleotides to achieve their full therapeutic potentials.

A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. Endcaps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. Endcaps containing either a terthiophene or a naphthalene tetracarboxylic acid dimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A•T base pair. Spacers containing only methylene (-CH 2 -) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

We have synthesized a series of short, self-complementary oligonucleotide sequences modified at their 5'- and/or 3'- termini with a lipophilic dodecane (C12); these systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5'-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (T(m)) by approximately 4-8 degrees C over their corresponding unmodified sequences. C12 functionalities added to both the 3'- and 5'-termini increased T(m) values by approximately 10-12 degrees C. The observed increases in T(m) correlated with greater duplex stabilities as determined by the free energy values (DeltaG) derived from T(m) plots. There is a greater degree of stabilization when C12 is positioned with a C.G base pair at the termini, and the stabilizing effect of lipophilic groups far exceeds the effect seen in adding an additional base pair to both ends of DNA. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and/or biological tools. These results suggest that the stability of short dsDNA sequences are improved by lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions.

We present an optimized synthetic strategy for the attachment of molecules to 5’-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH > 7.5 and releases them at pH < 6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.

The construction of nanomaterials from oligonucleotides by modular assembly invariably requires the use of branched nucleic acid architectures such as three- and four-way junctions (3WJ and 4WJ). We describe the stabilization of DNA 3WJ by using non-nucleotide lipophilic spacers to create a hydrophobic pocket within the junction space. Stabilization of nucleic acid junctions is of particular importance when constructing nanostructures in the "ultra-nano" size range (<20 nm) with shorter double-stranded regions. UV thermal melting studies show that lipophilic spacers strategically placed within the junction space significantly increased thermal stability. For a 3WJ with eight base pair arms, thermal stability was increased from 30.5 °C for the unmodified junction to a maximum stability of 55.0 °C. The stability of the junction can be modulated within this temperature range by using the appropriate combinations of spacers.