Name: development of cell-type specific anti-hiv gp120 aptamers for sirna delivery
Uploaded: 2011-06-23T12:00:00
Description: Watch this Scientific Journal Video about Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery at JoVE.com

We thank Britta Hoehn, Guihua Sun, Harris Soifer and Lisa Scherer for helpful discussions. This work was supported by grants from the National Institutes of Health AI29329 and HL07470 awarded to J.J.R. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: The CHO-EE and CHO-gp160 cell line; the pNL4-3 luc vector; HIV-1BaL gp120 from DAIDS, NIAID.

1. Preparation of the RNA library
<ol>
<li>The starting DNA library contained 50 nucleotides of random sequences and was synthesized by Integrated DNA Technologies (Coralville, Iowa). The single stranded DNA oligo library sequence is 5'- GGG AGG ACG ATG CGG - N50- CAG ACG ACT CGC CCG A - 3' (81 nt). The random region is flanked by constant regions, which include the T7 promoter for in vitro transcription and a 3' tag for RT-PCR. The 5' and 3' constant sequences are 5' &#x2013; TAA TAC GAC TCA CTA TA...

<ol>
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Aptamers are in vitro evolved nucleic acids that assume specific and stable three-dimensional shapes, thereby providing highly specific, tight binding to targeted molecules8. The low nanomolar binding affinity and exquisite specificity of aptamers to their targets make them versatile tools for diagnostics, in vivo imaging, and therapeutics9. With the advent of aptamer technology for targeted siRNA delivery it is now feasible to use the aptamer binding function for receptor mediated...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>MF-Millipore membrane filter</td>
			<td>Millipore</td>
			<td>HAWP01300</td>
			<td>Pore size 0.45 &mu;m</td>
		</tr>
		<tr>
			<td>Swinnex Filter holder</td>
			<td>Millipore</td>
			<td>SX0001300</td>
			<td>13 mm diameter</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>Microcon YM-30 column</td>
			<td>Millipore</td>
			<td>42410</td>
			<td>RNA concentration</td>
		</tr>
		<tr>
			<td>Bio-spin 30 column</td>
			<td>Bio-Rad</td>
			<td>732-6250</td>
			<td>RNA purification</td>
		</tr>
		<tr>
			<td>Taq PCR DNA polymerase</td>
			<td>Sigma-Aldrich</td>
			<td>D1806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ThermoScript RT-PCR system</td>
			<td>Invitrogen</td>
			<td>11146-024 </td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DuraScribe T7 transcription Kit</td>
			<td>EpiCentre</td>
			<td>DS010925</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dNTP for PCR</td>
			<td>Roche</td>
			<td>1 581 295</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ribonucleic acid, transfer from E.coli</td>
			<td>Sigma-Aldrich</td>
			<td>R1753</td>
			<td>tRNA competitor</td>
		</tr>
		<tr>
			<td>HIV-1Ba-L gp120 protein</td>
			<td>the AIDS Research and Reference Reagent Program</td>
			<td>4961</td>
			<td>Target protein</td>
		</tr>
		<tr>
			<td>Silencer siRNA labeling kit - Cy3</td>
			<td>Ambion</td>
			<td>1632</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acid phenol/chloroform 5/1 solution (pH 4.5)</td>
			<td>Ambion</td>
			<td>AM9720</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform/Isopropanol 24/1 solution</td>
			<td>Sigma</td>
			<td>C0549</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calf intestinal phosphatase (CIP)</td>
			<td>New England BioLab</td>
			<td>M0290L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England BioLab</td>
			<td>M0201L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10 901 393 001</td>
			<td>RNA precipitation</td>
		</tr>
		<tr>
			<td>Gamma-P32-ATP</td>
			<td>MP Biomedical</td>
			<td>013502002</td>
			<td>Radiactivity</td>
		</tr>
		<tr>
			<td>40% AccuGel 19:1</td>
			<td>National Diagnostics</td>
			<td>EC-850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10xTBE</td>
			<td>National Diagnostics</td>
			<td>EC-860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N,N,N,N'-Tetramethylethylenediamine (TMEMD)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-methioine sulfoximine</td>
			<td>Sigma-Aldrich</td>
			<td>M5379-250 mg</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI Media 1640</td>
			<td>Invitrogen</td>
			<td>11835-030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate solution, 7.5% w/v</td>
			<td>Invitrogen</td>
			<td>25080-094</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM) (10&#xF0B4;)</td>
			<td>Invitrogen</td>
			<td>11430-030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM non-essential amino acid (100&#xF0B4;)</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TA cloning kit with pCR 2.1 </td>
			<td>Invitrogen</td>
			<td>K2040-01</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

development of cell-type specific anti-hiv gp120 aptamers for sirna delivery

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).

Watch this Scientific Journal Video about Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery at JoVE.com

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

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