Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleotides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Use of a simple fractionation method to evaluate binding, internalization and intracellular distribution of oligonucleotides in vascular smooth muscle cells

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4 degrees C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25 kDa polyethylenimine (PEI) as a carrier. After 2.5 h of incubation at 37 degrees C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A large proportion (about 60%) of intracellular radioactivity remained associated with the endocytic compartment. Complexation with PEI completely changed intracellular distributions: the nuclear fraction increased to 70% for PS-ODN. The fractionation method proposed, facilitating study of the subcellular distribution of the ODN, could also be used under appropriate circumstances, to study variations in cytosolic ODN content.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Utilization of intrachain 4'-C-azidomethylthymidine for preparation of oligodeoxyribonucleotide conjugates by click chemistry in solution and on a solid support

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.     

Development of poly(ethylene glycol) conjugated lactoferrin for oral administration

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.     

Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature ( T m) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 degrees C for the 20-mer films to 53 degrees C for the 60-mer films. Further, an alternating trend in T m of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher T m (44-49 degrees C) than films with an outermost TC layer (ca. 38 degrees C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films.     

Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.     

Synthesis and fluorescence studies of multiple labeled oligonucleotides containing dansyl fluorophore covalently attached at 2'-terminus of cytidine via carbamate linkage

Synthesis of modified oligonucleotides in which the specific cytidine nucleoside analogues linked at 2'-OH position via a carbamate bond with an amino ethyl derivative of dansyl fluorophore is reported. For the multiple labeling of oligonucleotides, a strategy involving prelabeling at the monomeric level followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled probes has been described. The labeled monomer was phosphitylated using 2-cyanoethyl-N,N,N',N'-tetraisopropyl-phosphoramidite (Bis-reagent) and pyridiniumtrifluoro acetate (Py.TFA) as an activator. To ascertain the minimal number of labeled monomers required for a specific length of oligonucleotide for detection and also to assess the effect of carbamate linkage on hybridization, hexamer and 20-mer sequences were selected. Both were labeled with 1, 2, and 3 monomers at the 5'-end and hybridized with normal (unmodified) complementary sequences. As compared to midsequence or 3'-terminal labeling reported earlier, the 5'-terminal labeling has been found to have minimal contact-mediated quenching on duplex formation. This may be due to complementary deoxyguanosine (dG) rich oligonucleotide sequences or CG base pairs at a terminus that is known to yield stronger binding. This is one reason for selecting cytidine for labeling. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.     

Sensitive detection of unlabeled oligonucleotides using a paired surface plasma waves biosensor

Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides' relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization. PSPWB measures the demodulated amplitude of the heterodyne signal in real time. In the meantime, the ratio of the amplitudes between the detected output signal and reference can reduce the excess noise from the laser intensity fluctuation. Also, the common-path propagation of p and s waves cancels the common phase noise induced by temperature variation. Thus, a high signal-to-noise ratio (SNR) of the heterodyne signal is detected. The sequence specificity of oligonucleotide hybridization ensures that the platform is precisely discriminating between target and non-target oligonucleotides. Under optimized experimental conditions, the detected heterodyne signal increases linearly with the logarithm of the concentration of target oligonucleotide over the range 0.5-500 pM. The detection limit is 0.5 pM in this experiment. In addition, the non-target oligonucleotide at concentrations of 10 pM and 10nM generated signals only slightly higher than background, indicating the high selectivity and specificity of this method. Different length of perfectly matched oligonucleotide targets at 10-mer, 15-mer and 20-mer were identified at the concentration of 150 pM.     

Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice

Sepsis is associated with increased intestinal permeability, but mediators and mechanisms are not fully understood. We examined the role of interleukin (IL)-6 and IL-10 in sepsis-induced increase in intestinal permeability. Intestinal permeability was measured in IL-6 knockout (IL-6 -/-) and wild-type (IL-6 +/+) mice 16 h after induction of sepsis by cecal ligation and puncture or sham operation. In other experiments, mice or intestinal segments incubated in Ussing chambers were treated with IL-6 or IL-10. Intestinal permeability was assessed by determining the transmucosal transport of the 4.4-kDa marker fluorescein isothiocyanate conjugated dextran and the 40-kDa horseradish peroxidase. Intestinal permeability for both markers was increased in septic IL-6 +/+ mice but not in septic IL-6 -/- mice. Treatment of nonseptic mice or of intestinal segments in Ussing chambers with IL-6 did not influence intestinal permeability. Plasma IL-10 levels were increased in septic IL-6 -/- mice, and treatment of septic mice with IL-10 resulted in reduced intestinal permeability. Increased intestinal permeability during sepsis may be regulated by an interaction between IL-6 and IL-10. Treatment with IL-10 may prevent the increase in mucosal permeability during sepsis.     

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.     

Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V

A gene for T4 endonuclease V was constructed by joining chemically synthesized oligodeoxyribonucleotides and expressed efficiently in Escherichia coli under the control of the E. coli tryptophan promoter. Overproduced T4 endonuclease V, which can cleave thymine photodimers as well as the corresponding phosphodiester linkage of DNA, was used to investigate the precise mode of the reaction with single- or double-stranded synthetic DNA fragments containing a thymine photodimer. The substrates, three oligodeoxyribonucleotides, d(GCGGTTGGCG) (10-mer), d(CGAAGGTTGGAAGC) (14-mer), and d(CACGAAGGTTGGAAGCAC) (18-mer), were prepared by UV irradiation of the nascent oligonucleotides. These single-stranded oligonucleotides were cleaved by the enzyme with a concentration 100 times higher than that required for the corresponding duplexes. The Km values for the TT duplex (14- and 18-mer) were found to be on the order of 10(-8) M. Dissociation constants for the 14- and 18-mer duplexes were measured by a binding assay on a nitrocellulose filter and found to be 10(-9).     

Antisense oligonucleotides: Efficient synthesis of 2′-O-methoxyethyl phosphorothioate oligonucleotides using 4,5-dicyanoimidazole. Are these oligonucleotides comparable to those synthesized using 1H-tetrazole as coupling activator?

Multiple 2'-O-methoxyethyl modified phosphorothioate oligonucleotides of 18-20-mer in length were synthesized at various scales using 4,5-dicyanoimidazole (DCI) as coupling activator. Extensive synthetic, analytical (using ion-pair LC-MS), and in vivo pharmacological, toxicological studies showed that oligonucleotides made with DCI and 1H-tetrazole are chemically and biologically equivalent. This extensive study will help the oligonucleotide therapeutic industry to move from using a potentially explosive activator (1H-tetrazole) to a safe activator (DCI).     

Reducing the number of microlocations in oligonucleotide microchip matrices by the application of degenerate oligonucleotides

The application of degenerate oligonucleotides to DNA Sequencing by Hybridisation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerate oligonucleotides is regarded as an example of pooling methods that are suitable for various laboratory procedures requiring numerous samples to be assayed. As each DNA sequence coded by four letters (A, G, C, T) may be defined by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong-G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n4n -nucleotide sequences may be defined with the help of 2xn2sequences. In the place of the originally described microchip matrix composed of all possible unambiguous octanucleotides (4(8)=65 536) attached to the equal number of 65 536 microlocations a matrix composed of 512 microlocations containing 256 2(8)-degenerate octanucleotides is proposed. The matrix contains all 256 possible octanucleotides coded by W and S variations and all 256 possible octanucleotides coded by R and Y variations. The 512 256-degenerate octanucleotides allows to retrieve the same information as 65 536 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligonucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matrices, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonucleotides (ca. 4.3x10(9)).     

Ca2+-dependent changes in cyclic GMP levels are not correlated with opening and closing of the light-dependent permeability of toad photoreceptors

We have measured the levels of 3',5'-guanosine monophosphate (cyclic GMP) in isolated retinas from toad to investigate their correlation to the opening and closing of the light-dependent permeability of photoreceptors. When Ca2+-induced changes in cyclic GMP concentration are compared with the Ca2+-induced changes in the permeability of photoreceptor light-dependent channel, four quantitative dissimilarities are noted. First, when extracellular Ca2+ ([Ca2+]o) is reduced from normal physiological levels to between 10(-6) and 10(-7) M, the light-dependent permeability is increased, but cyclic GMP levels are not significantly changed. Second, when [Ca2+]o is increased from 1.8 to 20 mM, the light-dependent permeability is suppressed, but cyclic GMP levels are decreased by only 10-15%, about one-quarter the decrease that can be obtained with bright illumination. Third, when [Ca2+]o is increased from 10(-8) M to 20 mM, the light-dependent permeability is closed rapidly, but the cyclic GMP decrease is slow. Fourth, when [Ca2+]o is lowered to 10(-8) M, the sensitivity of the light-dependent permeability to steady illumination is decreased by three to four orders of magnitude, but the sensitivity of the light-dependent decrease in cyclic GMP is not significantly affected. These observations indicate that there is no simple correlation between cyclic GMP levels and the permeability of the light-dependent channels and that Ca2+ can affect the conductance in the absence of changes in cyclic GMP content.     

Complete protection of antisense oligonucleotides against serum nuclease degradation by an avidin-biotin system.

It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUT1 glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUT1 biotinylated oligonucleotide-avidin construct showing complete protection against serum 3'-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUT1 glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5'- or near 3'-biotinylated oligonucleotide (bio-DNA), and 5'- and 3'-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5'-32P]-5'-bio-DNA (> 95% at 30 min). Avidin partially protected this construct (approximately 31% of intact 21-mer oligo remained at 1 h). Similar results were obtained with the [3'-32P]-5'-bio-DNA; however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3'-exonuclease. Incubation of the [5'-32P]-3'-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1/2 approximately 13 min). Conversely, avidin totally protected this construct against the serum 3'-exonuclease. In conclusion, avidin fully protects antisense oligonucleotides biotinylated at the near 3'-terminus against serum 3'-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.