कई 2'Fluoro nanomole आत्मीयता के साथ एचआईवी 1Ba एल gp120 खिलाफ आरएनए aptamers एक शाही सेना पुस्तकालय से अलग कर रहे हैं द्वारा<em> इन विट्रो में</em> SELEX प्रक्रिया. एक नई दोहरी निरोधात्मक विरोधी gp120 समारोह कल्पना aptamer – siRNA बनाया है और प्रणालीगत थेरेपी विरोधी एचआईवी के लिए काफी वादा दिखाता है है.
The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery.
Herein, we firstly discuss the selection and identification of 2′-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).
Aptamers में इन विट्रो न्यूक्लिक एसिड होता है जो विशिष्ट और स्थिर तीन आयामी आकार को लगता है विकसित किया है, जिससे अत्यधिक विशिष्ट, लक्षित 8 अणुओं के लिए तंग बाध्यकारी प्रदान. कम nanomolar बंधन आत्मीयता और अप…
The authors have nothing to disclose.
हम Britta Hoehn, Guihua सन, हैरिस Soifer और उपयोगी विचार – विमर्श के लिए लिसा Scherer धन्यवाद. चो – ई और चो – gp160 सेल: यह काम NIH एड्स अनुसंधान और संदर्भ अभिकर्मक कार्यक्रम एड्स के प्रभाग, NIAID, एनआईएच के माध्यम से प्राप्त थे AI29329 स्वास्थ्य और HL07470 निम्नलिखित अभिकर्मकों JJR करने के लिए सम्मानित किया के राष्ट्रीय संस्थानों से अनुदान द्वारा समर्थित किया गया था रेखा; pNL4-3 ल्यूक वेक्टर, एचआईवी -1 से gp120 बाल DAIDS, NIAID.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
MF-Millipore membrane filter | Millipore | HAWP01300 | Pore size 0.45 μm |
Swinnex Filter holder | Millipore | SX0001300 | 13 mm diameter |
QIAquick Gel Extraction Kit | QIAGEN | 28706 | DNA purification |
Microcon YM-30 column | Millipore | 42410 | RNA concentration |
Bio-spin 30 column | Bio-Rad | 732-6250 | RNA purification |
Taq PCR DNA polymerase | Sigma-Aldrich | D1806 | |
ThermoScript RT-PCR system | Invitrogen | 11146-024 | |
DuraScribe T7 transcription Kit | EpiCentre | DS010925 | |
dNTP for PCR | Roche | 1 581 295 | |
Ribonucleic acid, transfer from E.coli | Sigma-Aldrich | R1753 | tRNA competitor |
HIV-1Ba-L gp120 protein | the AIDS Research and Reference Reagent Program | 4961 | Target protein |
Silencer siRNA labeling kit – Cy3 | Ambion | 1632 | |
Acid phenol/chloroform 5/1 solution (pH 4.5) | Ambion | AM9720 | |
Chloroform/Isopropanol 24/1 solution | Sigma | C0549 | |
Calf intestinal phosphatase (CIP) | New England BioLab | M0290L | |
T4 polynucleotide kinase | New England BioLab | M0201L | |
Glycogen | Roche | 10 901 393 001 | RNA precipitation |
Gamma-P32-ATP | MP Biomedical | 013502002 | Radiactivity |
40% AccuGel 19:1 | National Diagnostics | EC-850 | |
10xTBE | National Diagnostics | EC-860 | |
N,N,N,N’-Tetramethylethylenediamine (TMEMD) | Sigma-Aldrich | T9281 | |
Ammonium persulfate (APS) | Sigma-Aldrich | A3678 | |
L-methioine sulfoximine | Sigma-Aldrich | M5379-250 mg | |
RPMI Media 1640 | Invitrogen | 11835-030 | |
Sodium Bicarbonate solution, 7.5% w/v | Invitrogen | 25080-094 | |
Minimum Essential Medium (MEM) (10) | Invitrogen | 11430-030 | |
MEM non-essential amino acid (100) | Invitrogen | 11140-050 | |
TA cloning kit with pCR 2.1 | Invitrogen | K2040-01 |