Attenuating HIV Tat/TAR-mediated protein expression by exploring the side chain length of positively charged residues.

RNA is a drug target involved in diverse cellular functions and viral processes. Molecules that inhibit the HIV TAR RNA-Tat protein interaction may attenuate Tat/TAR-dependent protein expression and potentially serve as anti-HIV therapeutics. By incorporating positively charged residues with mixed side chain lengths, we designed peptides that bind TAR RNA with enhanced intracellular activity. Tat-derived peptides that were individually substituted with positively charged residues with varying side chain lengths were evaluated for TAR RNA binding. Positively charged residues with different side chain lengths were incorporated at each Arg and Lys position in the Tat-derived peptide to enhance TAR RNA binding. The resulting peptides showed enhanced TAR RNA binding affinity, cellular uptake, nuclear localization, proteolytic resistance, and inhibition of intracellular Tat/TAR-dependent protein expression compared to the parent Tat-derived peptide with no cytotoxicity. Apparently, the enhanced inhibition of protein expression by these peptides was not determined by RNA binding affinity, but by proteolytic resistance. Despite the high TAR binding affinity, a higher binding specificity would be necessary for practical purposes. Importantly, altering the positively charged residue side chain length should be a viable strategy to generate potentially useful RNA-targeting bioactive molecules.

Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Evaluation of the safety and brain-related tissues distribution characteristics of TAT-HaFGF via intranasal administration.

Disabilities triggered by neurodegeneration mainly result in mortality in the elderly, and patients with neurodegenerative disease also display deficits in olfactory function. Therefore drug distribution to the brain through intranasal administration has become one of the most difficult challenges in the treatment of central nervous system (CNS) diseases. TAT-human acidic fibroblast growth factor (HaFGF) is a new fused protein retaining the neuroprotective activities of HaFGF, and is a promising prospect in the treatment of neurodegenerative diseases. TAT (a cell-penetrating peptide) contains a high relative abundance of positively charged amino acids such as lysine and arginine, which have a powerful attraction to the negatively charge on the nasal epithelial membrane. The present study focused on the evaluation of the safety and absorption characteristics of TAT-HaFGF following intranasal administration. After TAT-HaFGF intranasal administration (100, 300, 600 µg/kg) for 5 weeks, hematoxylin-eosin (HE) staining showed no pathology in any of the investigated tissues and organs. The expression of olfactory marker protein (OMP) was observed with immunohistochemical staining, which showed no altered expression in the sensory neurons of the nasal epithelium. Nasal ciliotoxicity studies carried out using an in situ palate model and optical microscope showed that TAT-HaFGF had no nasal ciliotoxicity. The distribution of the TAT-HaFGF following intranasal administration was assessed using a radioisotopic tracing method. Radioactivity was observed in the brain after 15 min. This became stronger at 30 min and weaker at 1 h. All of the results confirmed the in vivo safety of TAT-HaFGF via intranasal administration.

Transdermal absorption and stability enhancement of salmon calcitonin by Tat peptide.

CONTEXT: Highly organized structure of stratum corneum (SC) is the major barrier of the delivery of macromolecules such as proteins and peptides across the skin. Recently, cell penetrating peptides (CPPs) such as HIV1-trans-activating transcriptional (Tat) have been used to enhance the topical delivery of proteins and peptides. OBJECTIVE: This study aimed to enhance the transdermal absorption and chemical stability of salmon calcitonin (sCT) by co-incubation with Tat. MATERIALS AND METHODS: Tat-sCT mixture at 1:1 molar ratio was prepared. Transdermal absorption and chemical stability of the mixture was evaluated in comparing with free sCT. RESULTS: Tat-sCT mixture gave higher cumulative amounts and fluxes of sCT than free sCT. The maximum percentage of sCT of 58.36 ± 12.33% permeated into the receiving chamber was found in Tat-sCT mixture at 6 h which was 3.50 folds of free sCT. Tat-sCT mixture demonstrated better sCT stability than sCT solution after 1 month storage at 4°C, 25°C and 45°C. DISCUSSION: The positively-charged arginine groups in Tat might be responsible for the binding of peptide complexes to negatively charged cell surfaces by electrostatic interactions and also the translocation of sCT through the excised skin. CONCLUSION: This study demonstrated the enhancements of transdermal absorption and stability of sCT by Tat peptide with potential for further application in transdermal delivery of other therapeutic peptides.

Synergistic effects of cell-penetrating peptide Tat and fusogenic peptide HA2-enhanced cellular internalization and gene transduction of organosilica nanoparticles.

The nonviral gene delivery system is an attractive alternative to cancer therapy. A new kind of gelatin-silica nanoparticles (GSNPs) was developed through a two-step sol-gel procedure. To improve the transfection efficacy, GSNPs modified with different fusion peptides (Tat, HA2, R8, Tat/HA2, and Tat/R8) were prepared for particle size, zeta potential, cellular uptake, hemolysis activity at physiological pH (7.0) or acidic pH (5.0), and condensation of plasmid DNA. The results suggest that the sizes and zeta potentials of GS-peptide conjugates were 147 - 161 nm and 19 - 33 mV, respectively; GS-peptide conjugates exhibited low cytotoxicity; the plasmid DNA was readily entrapped at a GS-peptide/pDNA weight ratio of 50 - 200. The in vitro and in vivo studies demonstrated that the synergistic effects of cell-penetrating peptide Tat and fusogenic peptide HA2 could promote the efficient cellular internalization, endosome escape, and nucleus targeting, hence delivering the therapeutic nucleic acid efficiently.

Human serum albumin fusion protein as therapeutics for targeting amyloid beta in Alzheimer's diseases.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.

[Potential of cell penetrating peptides for cell drug delivery]. / Administration de molécules actives dans les cellules: le potentiel des peptides de pénétration cellulaire.

The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes… This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions.

[Pharmacokinetics of a fusion protein for human acidic fibroblast growth factor and transcriptional activator protein in rat and its penetration across blood-brain barrier].

This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.

[Study of TAT-PEG loaded magnetic liposomes crossing blood spinal cord barrier after spinal cord injury in rats].

OBJECTIVE: To evaluate the ability of a kind of novel magnetic liposomes modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) to cross the blood spinal cord barrier (BSCB) so as to demonstrate whether or not they can accumulate at the lesions of injured spinal cord. METHODS: The novel liposomes were made through reverse-phase evaporation method modified with polyethylene glycol (PEG) and transactivating-transduction protein (TAT) with an iron core. Thirty-six Wistar rats subject to spinal cord injury (SCI) at T10 were randomly divided into three groups (Groups I, II and III). The rats of Group III were injected with TAT-PEG loaded magnetic liposomes (4.55 mg/kg). The rats of GroupII received an injection of the equivalent PEG loaded magnetic liposomes while those of control group (GroupI) the equivalent normal saline. The accumulation of liposomes was observed by MRI (magnetic resonance imaging), Prussian blue staining, electron microscope and flame atomic absorption spectrophotometer. RESULTS: This kind of TAT-PEG loaded magnetic liposomes could cross the BSCB and enter into the cells around the injured tissue. A low signal of T2WI on MRI could also be found in Group III. The results of flame atomic absorption spectrophotometer showed that the iron content accumulated around the lesion site in Group III was obviously higher than the other two groups (P < 0.05). CONCLUSION: The TAT-PEG loaded magnetic liposomes may be employed as one kind of novel drug carrier to cross the BSCB and accumulate at tissue cells of spinal cord. It is likely to become a new therapy for SCI.

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells.

BACKGROUND: Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes. METHODS: Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy. RESULTS: Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi. CONCLUSIONS: Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved. GENERAL SIGNIFICANCE: These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking.

The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.

Selective inhibition of ErbB2-overexpressing breast cancer in vivo by a novel TAT-based ErbB2-targeting signal transducers and activators of transcription 3-blocking peptide.

ErbB2 is an excellent target for cancer therapies. Unfortunately, the outcome of current therapies for ErbB2-positive breast cancers remains unsatisfying due to resistance and side effects. New therapies for ErbB2-overexpressing breast cancers continue to be in great need. Peptide therapy using cell-penetrating peptides (CPP) as peptide carriers is promising because the internalization is highly efficient, and the cargoes delivered can be bioactive. However, the major obstacle in using these powerful CPPs for therapy is their lack of specificity. Here, we sought to develop a peptide carrier that could introduce therapeutics specifically to ErbB2-overexpressing breast cancer cells. By modifying the HIV TAT-derived CPP and conjugating anti-HER-2/neu peptide mimetic (AHNP), we developed the peptide carrier (P3-AHNP) that specifically targeted ErbB2-overexpressing breast cancer cells in vitro and in vivo. A signal transducers and activators of transcription 3 (STAT3)-inhibiting peptide conjugated to this peptide carrier (P3-AHNP-STAT3BP) was delivered more efficiently into ErbB2-overexpressing than ErbB2 low-expressing cancer cells in vitro and successfully decreased STAT3 binding to STAT3-interacting DNA sequence. P3-AHNP-STAT3BP inhibited cell growth in vitro, with ErbB2-overexpressing 435.eB breast cancer cells being more sensitive to the treatment than the ErbB2 low-expressing MDA-MB-435 cells. Compared with ErbB2 low-expressing MDA-MB-435 xenografts, i.p. injected P3-AHNP-STAT3BP preferentially accumulated in 435.eB xenografts, which led to more reduction of proliferation and increased apoptosis and targeted inhibition of tumor growth. This novel peptide delivery system provided a sound basis for the future development of safe and effective new-generation therapeutics to cancer-specific molecular targets.

Modulation of nuclear internalization of Tat peptides by fluorescent dyes and receptor-avid peptides.

The nuclear internalization of biomolecules by Tat peptide provides a mechanism to deliver drugs to cells. However, translocation of molecular imaging probes to the nucleus may induce undesirable mutagenesis. To assess the feasibility of retaining its cell permeating effect without nuclear translocation, Tat-peptide was conjugated with a somatostatin receptor (STR)-avid ligand (Oct) and labeled with fluorescent dyes. The results show that Tat-Oct-5-FAM (fluorescein 5'-carboxylic acid) remained in the cytoplasm of STR-positive AR42J cells. Co-incubation of Tat-Oct-5-FAM with ATP induced nuclear translocation. These data suggest that both dye and Oct-STR endocytosis complex could modulate nuclear internalization of Tat peptides.

Internalization of novel non-viral vector TAT-streptavidin into human cells.

BACKGROUND: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47-57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. RESULTS: By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs. CONCLUSION: This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells.

Pharmacokinetics of 125I-GST-TatdMt, a recombinant fusion protein possessing potent anti-obesity activity, after intravenous, nasal, oral, and subcutaneous administration.

This study first reports the absorption kinetics of GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in rats after nasal, s.c., and p.o. administration. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. The radioiodinated 125I-GST-TatdMt was administered to rats by nasal, s.c., and oral routes at doses of 7.3 microg (420.7 nCi), 146.5 microg (8413.8 nCi), and 146.5 microg (8413.8 nCi), respectively. For the determination of absolute bioavailability, 125I-GST-TatdMt was also given to rats by i.v. injection (73.2 microg, 4206.9 nCi). Following administration by extravascular routes, the systemic absorption of radioactivity was prolonged, with Cmax being attained within 4.2-8.0 h. The absolute bioavailability calculated as dose-normalized AUC(extravascular)/AUC(i.v.) was 98.0, 75.8, and 87.1% after nasal, s.c., and oral administration, respectively. The majority of administered radioactivity was excreted in urine (57.5-64.7%), with fecal excretion being less (2.5-12.7%). The distribution of 125I-GST-TatdMt to various tissues was also determined at 4 and 72 h after s.c. injection. The findings of this study suggest that this protein may be absorbed into the systemic circulation when given by extravascular administration.

Cellular penetration and nuclear importation properties of 111In-labeled and 123I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells.

INTRODUCTION: Our objective was to compare the cell penetration and nuclear importation properties of 111In-labeled and 123I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. METHODS: [111In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO4(-)-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [111In]anti-mIgG-tat IC and [123I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH4Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [111In]anti-mIgG-tat were measured. RESULTS: [111In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [123I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [111In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [111In]anti-mIgG-tat were inhibited by excess tat peptides and NH4Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for 123I-labeled than for 111In-labeled anti-mIgG-tat ICs. CONCLUSION: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of 111In-labeled and 123I-labeled anti-mIgG antibodies with BT-474 human BC cells. 111In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for imaging and/or radiotherapeutic applications.

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

Pharmacokinetics of GST-TatdMt, a recombinant fusion protein possessing potent anti-obesity activity, in mice.

This study examined the absorption and pharmacokinetic disposition of 125I-GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in mice after vascular and extravascular administration. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. 125I-GST-TatdMt was administered to mice by i.v., i.p. and oral administration at doses of 652.7 nCi (102.3 microg). Upon i.v. injection, the average terminal elimination half-life (t1/2,lambdaz), AUC and AUMC were 6.4 h, 318.2 nCixh/mL and 2518 nCixh2/ mL, respectively. The highest radioactivity was observed in lung followed by liver, spleen, heart and kidney. The t1/2lambdaz values obtained from i.v., i.p., and oral administration were comparable from each other (range 5.8-6.4 h). The absolute bioavailability of 125I-GST-TatdMt was 42.8% and 60.5% after p.o. and i.p. administration, respectively. Given the cell-penetrating nature, 125I-GST-TatdMt may be absorbed into the systemic circulation to a relatively high extent after extravascular administration.

Selective cell uptake of modified Tat peptide-fluorophore conjugates in rat retina in ex vivo and in vivo models.

PURPOSE: To determine the pattern of retinal uptake of modified Tat peptide-fluorophore conjugates in the rat after ex vivo application and intravitreal injection. METHODS: Modified Tat peptide (RKKRRORRRGC) was conjugated at the C terminus to Alexa Fluor 594 to enable visualization of uptake. In the ex vivo model, posterior segments were incubated for up to 120 minutes in peptide solution. In the in vivo model, intravitreal injections of 5 microL peptide solution were performed in anesthetized rats, which were then euthanatized from 1 hour to 7 days after injection. Retinal and optic nerve paraffin sections were examined for fluorescent labeling. Immunohistochemistry for retinal cell markers was performed to identify cell types exhibiting uptake. RESULTS: The pattern of labeling seen in retinal sections was highly similar for the ex vivo and in vivo experiments, with specific uptake by retinal ganglion cells (RGCs) and by a subset of inner nuclear layer cells. The pattern of labeling remained specific even at the later time points. In the in vivo model, fluorescence was also noted in the nerve fiber layer and anterior optic nerve, extending posteriorly along the optic nerve at later time points. CONCLUSIONS: A specific pattern of uptake for modified Tat peptides was consistently seen in the rodent retina. Given the preferential uptake of these peptides by RGCs and the potential to conjugate diverse moieties, modified Tat peptides may be useful for delivery of therapeutic agents or molecular imaging probes to RGCs.

Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics.

The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.