In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection

Abstract

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

In Vitro Selection of RNA Aptamers That Bind to Cell Adhesion Receptors of Trypanosoma cruzi and Inhibit Cell Invasion

“…The biological activity of such aptamers have been shown in in vitro assays where RNA antagonists inhibited selectin-dependent adhesion (17,24). Wang et al (25) selected RNA aptamers that bind to infectious human cytomegalovirus and inhibit viral infection in vitro, showing the feasibility of the SELEX technique for the evolution of novel compounds that protect cells against infection by pathogens. Furthermore, combinatorially synthesized nuclease-resistant RNA and DNA ligands are promising candidates for use in pharmaceutical and therapeutic applications (cf.…”

A selection system for identifying accessible sites in target RNAs

“…Experimental approaches for identification of cleavable sites offer clear advantages+ Lieber and Strauss (1995) constructed a library of hammerhead ribozymes+ These library ribozymes were targeted to a preselected triplet, and contained randomized sequences in the annealing arms, which allowed the screening of accessible sites in the target-RNA molecule+ In this case, the selected ribozymes cleaved an in vitro transcript efficiently and inhibited gene expression in cell culture; one selected ribozyme was successfully used for inhibition of growth-hormone expression in mice (Lieber & Kay, 1996)+ In a different approach, Birikh et al+ (1997b) used a completely randomized oligonucleotide (dN 10 ) in conjunction with RNase H to map sites that are accessible for oligonucleotide binding in an RNA transcript: the best ribozymes generated in this fashion was 150-fold more active than the most efficient ribozymes designed on the basis of the mFold program (Zuker & Stiegler, 1981)+ As a remarkably powerful tool, the systematic evolution of ligands by exponential enrichment (SELEX; Ellington & Szostak, 1990;Tuerk & Gold, 1990) has been used to isolate oligonucleotide sequences, socalled aptamers, with the capacity to recognize virtually any class of target molecules with high affinity and specificity, such as organic dyes, amino acids, biological cofactors, antibiotics, peptides and proteins, or even whole viruses and protozoan organisms (Pan et al+, 1995;Eaton, 1997;Osborne & Ellington, 1997;Bell et al+, 1998;Gal et al+, 1998;Kraus et al+, 1998;Yang et al+, 1998;Homann & Goringer, 1999;Wang et al+, 2000)+ Here, we have extended the SELEX method to isolate ribozyme-guide RNAs (Fig+ 2), which can aid in the location of optimal cleavage sites within any targeted RNA, based upon Watson-Crick base pairing between random guide RNAs and target RNAs under physiologic conditions (Fig+ 1B)+ Using long (partial and/or full-length) transcripts of human hepatitis B virus RNA (HBV, 917 nt long), human RNA polymerase I (the hRPA39 subunit of Pol I, 1,039 nt long) and mouse tumor suppressor gene "phosphatase and tensin homologue deleted on chromosome ten" (PTEN, 1,213 nt), we demonstrate that highly effective ribozymes can be engineered by systematically isolating a pool of ribozyme-guide RNA from a large library of random guide RNA sequences+ Ribozymes targeted to these library-selected sites efficiently cleaved long transcripts in vitro: the best showed a K cat /K m ϭ 1+0 ϫ 10 6 (M Ϫ1 min Ϫ1 ), whereas others generally showed K cat /K m values of 0+6 ϫ 10 6 (M Ϫ1 min Ϫ1 )+ Library-selected ribozymes targeted to HBV effectively inhibited viral FIGURE 1. Schematic representation of the Random Selection Library+ A: Diagram of a hammerhead ribozyme, showing a catalytic core, a random 6-nt 59-flanking region and a 9-nt 39-flanking region+ Arrowhe...…”

Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation

“…To identify RNA molecules capable of binding with high affinity to the Us11 protein, we employed an in vitro selection technique that has been used previously to successfully isolate RNA ligands that bind to a variety of relatively simple and complex molecular structures (2,4,11,13,45,51,55). This methodology allowed us to amplify specific RNA ligands from a combinatorial library consisting of molecules with a randomized central region.…”

“…An oligonucleotide library that contains an internal randomized stretch of 40 nucleotides flanked by a 5Ј invariant region that contains the T7 promoter and a 3Ј invariant region was a gift from Fenyong Liu (University of California-Berkeley) and is described in reference 51. The library was amplified (25 cycles of 94°C for 1 min, 47°C for 1 min, and 72°C for 1 min; on the final cycle, a 10-min extension was performed) with Taq DNA polymerase using the primers JH1031 and JH1052 (51). Following purification of the PCR product on a 2% agarose gel, an in vitro RNA synthesis reaction was assembled with T7 RNA polymerase (NEB) using buffer supplied by the manufacturer supplemented with 0.5 mM nucleoside triphosphates, 20 U of RNasin (Promega), and 30 Ci of [␣ 32 P]CTP (3,000 Ci/mmol).…”