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

Summary

Several 2’-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.

Abstract

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).

Protocol

1. Preparation of the RNA library

1. 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' – TAA TAC GAC TCA CTA TAG GGA GGA CGA TGC GG - 3' (32 mer) and 5' -TCG GGC GAG TCG TCT G - 3' (16 mer), respectively. Make stock solution with water and store in aliquots at -20°C.<....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MF-Millipore membrane filter	EMD Millipore	HAWP01300	Pore size 0.45 μm
Swinnex Filter holder	EMD Millipore	SX0001300	13 mm diameter
QIAquick Gel Extraction Kit	Qiagen	28706	DNA purification
Microcon YM-30 column	EMD 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 Biotechnologies	DS010925
dNTP for PCR	Roche Group	1 581 295
Ribonucleic acid, transfer from E.coli	Sigma-Aldrich	R1753	tRNA competitor
HIV-1Ba-L gp120 protein	National Institutes of Health	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-Aldrich	C0549
Calf intestinal phosphatase (CIP)	New England Biolabs	M0290L
T4 polynucleotide kinase	New England Biolabs	M0201L
Glycogen	Roche Group	10 901 393 001	RNA precipitation
Gamma-P32-ATP	MP Biomedicals	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