Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo

Abstract

Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.

Figure 1

Identification of accessible regions on the cyclin transcripts with RNase H/dN₁₂ (A) Schematic representation of RNase H digestion of end-labelled mRNA. (B) Autoradiographs showing gel analysis of the cleaved products of 5′-end-labelled mRNA with RNase H at various dN₁₂ concentrations. The transcripts were titrated against various concentrations of the oligomer library, ranging from 10 to 500 pmol/reaction (lane 0, native transcript; lane 1, plus RNase H; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 50 pmol; lane 5, 100 pmol; lane 6, 250 pmol; lane 7, 500 pmol). Each reaction contained ∼3 × 10⁹ transcript molecules. The dN₁₂ library contains 1.67 × 10⁷ different 12mers. There are ∼1.8 × 10⁷ molecules of each oligonucleotide in 500 pmol, which resulted in almost complete cutting of the full-length transcripts. How can this number of oligonucleotides mediate cleavage of 3 × 10⁹ target molecules? Since regions of complementarity between RNA and an oligonucleotide of only 4 nt can recruit RNase H activity (25), the oligonucleotides in the library that were only partially complementary to the accessible sites would also be expected to participate in RNase H-mediated cutting of the transcript. In addition, each oligonucleotide can participate more than once in successive reactions.

Figure 2

The effects on binding capacity of sequences and lengths of antisense oligonculeotides determined on scanning arrays. (A) Schematic representation of a scanning array. The sequences given in the cartoon correspond to cyclin B5. (B) Each of the three panels shows an image obtained after hybridisation of the radiolabelled transcripts to the corresponding scanning array. The histograms, obtained with the program xvseq, represent binding of the 15mer oligonucleotides to the transcript on the array. Histograms corresponding to only half of the arrays are shown. The sequences of the antisense oligonucleotides used in various assays are shown on the histograms and underlined in the 120 nt sequence of the cyclin mRNAs.

Figure 3

Folded structures of the three cyclin mRNAs obtained using mfold. Only the relevant sections of the folds are shown. The sequences analysed on arrays are marked (start and end are indicated). The binding sites of the oligonucleotides are marked with bold lines on each structure. The grey line in the B1 mRNA fold represents overlap between oligonucleotides B1-2 and B1-3.

Figure 4

Effects of oligonucleotides on cell-free protein synthesis in frog egg extract. (A) The pattern of radioactive proteins displayed on SDS–PAGE after addition of 1 µM of the indicated oligonucleotides. The asterisk indicates an unidentified high molecular weight translation product whose synthesis is particularly sensitive to non-specific inhibition by the added DNA. (B) The radiolabelled immunoprecipitates obtained with the indicated antisera in response to the various oligonucleotides.

Figure 5

(A) Designation, location and hybridisation intensities of the oligonucleotides tested for their effects on translation in (B), which shows both the total pattern of protein synthesis and the specific synthesis of B5 using immunoprecipitation and autoradiography. (C) Titration of selected oligonucleotides from 2 to 0.125 µM in five 2-fold dilution steps as indicated by the wedges. Note that oligonucleotides c, f and o cover the same region of the mRNA, but differ in length and hybridisation intensity as indicated. Oligonucleotide d comes from a poorly scoring region of the array.

Figure 6

Cleavage of cyclin B5 mRNA by antisense 15mers injected into intact Xenopus oocytes. (A) The location of four antisense oligonucleotides on the ∼350 nt probe and approximate sizes of the 3′-end cut products (drawings only roughly to scale). (B) The RNase protection map for cyclin B5 mRNA in response to antisense-mediated cleavage produced by 25, 50 and 100 ng each oligonucleotide as indicated by the wedges. Cyclin B3 was used as a negative control and the products of cleavage are indicated on the right side of the figure. The additional fainter bands seen in the B5-3 and B5-4 lanes possibly represent further degradation products of the B5 mRNA after cleavage with antisense oligonucleotides, since the oligonucleotides do not have sufficient complementarity in the neighbouring regions (data not shown) for secondary binding to produce faint bands of these sizes.

Figure 7

Time course of cleavage of cyclin B1 mRNA and effects on the levels of cyclin B1 protein in response to oligonucleotide B1-3. (A) Cleavage of cyclin B1 mRNA as measured by RNase protection mapping at the indicated times after injection of the DNA into progesterone-treated Xenopus oocytes. (B) Immunoblots of cyclin B1 and Cdc2 (loading control) in progesterone-treated oocytes at the time of GVBD (0) or 1 h later after injection of water or oligonucleotide solution as indicated or addition of 100 µg/ml cycloheximide (CHX) to the medium.