Method for specifically altering the nucleotide sequence of RNA

A method is provided for specifically altering the nucleotide sequence of an RNA molecule. As performed in vitro the method comprises: (1) selecting a target sequence in an RNA molecule; (3) hybridizing the target sequence with a complementary nucleic acid, either DNA or RNA, thereby forming a double-stranded structure; (4) incubating the hybrid molecule with a cellular extract which contains components capable of specifically converting U to C in an RNA molecule, when the U is disposed within a double-stranded structure. Incubation of the hybrid molecule with cellular extracts comprising the RNA editing component results in a specific alteration of the nucleotide sequence of the RNA at the target sequence. The method of the invention may also be applied in vivo. Complementary nucleic acids are introduced into cells and hybridized to the target sequence to form a double-stranded structure. The RNA editing components already present in the cell may then specifically convert U to C in the target sequence resulting in an alteration in the target RNA molecule.

FIELD OF THE INVENTION 
This invention relates to the field of RNA therapy. In particular, a method 
is provided for editing an RNA molecule by selectively converting uridine 
to cytidine in a target RNA sequence disposed within the RNA molecule. 
BACKGROUND OF THE INVENTION 
Recent advances in recombinant DNA technology have brought the substantial 
promise of gene therapy nearer to realization. Techniques for specifically 
altering the nucleotide sequence of a DNA molecule have been a 
particularly valuable development. These techniques enable a DNA molecule 
to be tailored to provide a specific selected effect, when provided as 
gene therapy. 
An alternative to gene therapy is to manipulate gene expression at the RNA 
level. Targeting RNA has several advantages not inherent in gene 
manipulation at the DNA level. For example, RNA therapy can be targeted to 
cells and tissues where gene expression is actually occurring, i.e., cells 
in which mRNA encoding a specific gene product is being actively 
transcribed and translated. Additionally, in certain situations, RNA is a 
good target for gene therapy because RNA is an intermediate in reverse 
transcription, whereby certain viral DNA genomes are produced. 
Already, RNA-targeted gene therapy has been accomplished through the use of 
anti-sense RNA molecules (RNA or DNA) designed specifically to block the 
translation of a target RNA molecule into protein. Assuming knowledge of 
the nature of the target mRNA molecule, complementary oligonucleotides are 
created to bind specifically to regions of the RNA molecule critical for 
selected functions of the molecule, such as maintenance of secondary 
structure or translation, thereby disrupting expression of the gene 
product encoded by the RNA. 
Another type of RNA-targeted gene therapy involves the use of 
sequence-specific endoribonucleases, known as ribozymes, to achieve 
cleavage and inactivation of gene transcripts in vivo. According to this 
strategy, assuming knowledge of the mRNA transcribed by the gene, 
ribozymes can be synthesized which hybridize specifically to a 
predetermined sequence and cleave the RNA molecule at a specified site in 
the target sequence, so as to inhibit production of the gene product by 
destroying its messenger. 
Both ribozymes and anti-sense molecules are capable of disrupting gene 
expression at the RNA level, ribozymes by targeted destruction of the 
specific RNA, and anti-sense molecules by disrupting secondary structure 
or blocking translation. 
Although inhibiting gene expression is certainly a valuable mode of RNA 
therapy, it would be even better to have RNA-targeted methods for 
enhancing gene expression (by increasing the stability or translation 
efficiency of an RNA molecule) or altering the subsequently produced gene 
product. Such methods are available for DNA-targeted gene therapy (e.g., 
in vitro mutagenesis), but not for RNA-targeted gene therapy. Clearly, a 
method for altering the nucleic acid sequence of an RNA molecule could 
provide many new therapeutic methods by combining the advantages inherent 
in specifically altering DNA with the advantages of targeting RNA. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a method is provided for 
specifically altering the nucleotide sequence of an existing RNA molecule. 
This alteration is sometimes referred to herein as "RNA editing," and is 
accomplished by converting uridine to cytosine in a single stranded target 
sequence disposed within the RNA molecule. The target sequence comprises 
an editable segment which has at least one uridine (the "target 
nucleotide") disposed therein, and flanking segments, several nucleotides 
in length, which abut the editable segment on either side of the editable 
segment. 
According to one aspect of the invention, adapted for in vitro use, the 
method comprises providing a single stranded nucleic acid (DNA or RNA) 
which has a sequence complementary to the target sequence. The RNA editing 
function is provided by a nuclear extract or a whole cell extract having a 
significant amount of nuclear material. In accordance with the present 
invention, such extracts have been found to possess one or more RNA 
editing components capable of converting uridine to cytidine when the 
uridine is disposed within a double-stranded nucleic acid structure at a 
position several nucleotides inward from each terminus of the 
double-stranded structure. 
According to the method, the RNA molecule is hybridized with the 
complementary nucleic acid, thereby forming a hybrid molecule having a 
double-stranded structure made up of the target sequence and the 
complementary nucleic acid. The hybrid molecule is then treated with the 
nuclear extract having the RNA editing component, under conditions which 
promote conversion of uridine to cytidine when the uridine is disposed 
within a double-stranded nucleic acid structure. The RNA editing component 
then exerts its activity, thereby converting the uridines disposed within 
the editable segment to cytidines. In this way, the RNA molecule is 
specifically edited by converting uridine to cytidines in the editable 
segment of the target sequence disposed within the RNA molecule. 
With prior knowledge of the sequence of the selected RNA molecule, any 
target sequence within that molecule may be selected for editing. 
Nuclear extracts having the RNA editing component may be obtained from a 
variety of sources. In a preferred embodiment, whole cell extracts of 
Drosophila embryos are utilized in the editing process. 
At least one uridine is disposed within the editable segment. The editable 
segment may comprise additional uridines, as long as the flanking segments 
are several nucleotides in length. 
According to another aspect of the present invention, the complementary 
single stranded nucleic acid, which may be composed either of DNA or RNA, 
may be utilized in vivo. Cultured cells may be transfected with 
complementary nucleic acids of the invention. For intact multicellular 
organisms (e.g., mammals), in vivo delivery of the complementary nucleic 
acids to cells and tissues may be accomplished by methods such as those 
used for delivery of anti-sense or ribozyme molecules. For example, 
oligonucleotides may be delivered by targeted liposomes. Alternatively, 
target cells may be transformed with DNA molecules that, upon DNA-directed 
RNA transcription, form the appropriate complementary RNA to hybridize in 
vivo with the target RNA sequence. 
The RNA editing method of the invention provides notable advantages over 
RNA therapy methods currently available. Similar to anti-sense and 
ribozyme technologies, the method of the invention can be used to disrupt 
the functionality of an RNA molecule. But, whereas anti-sense and 
ribozymes are limited only to disruption, the method of the invention can 
also be used to re-direct RNA functionality, e.g., to enhance the 
efficiency of an RNA molecule or to alter the structure of the protein 
produced by an RNA. 
DETAILED DESCRIPTION OF THE INVENTION 
The following words and phrases are defined, for reference in describing 
the invention, as follows: 
1. RNA editing: refers to a specific alteration of the nucleotide sequence 
of an RNA molecule. In practicing the present invention, the nucleic acid 
sequence of a pre-determined target sequence of a selected RNA molecule is 
altered. The alteration comprises converting U in the editable segment of 
the target sequence to C, as described more fully below. The uridines 
targeted for editing are sometimes referred to herein as "target 
nucleotides." 
2. Nuclear extract: refers to an extract of soluble components of isolated 
nuclei, or an extract of soluble components of whole cells of a type 
comprising a high percentage of nuclear material, or an extract of soluble 
components of whole cells, wherein the extract is prepared so as to be 
enriched in components normally residing in the cell nucleus. 
3. RNA editing component: refers to one or more components in a nuclear 
extract, as defined above, capable of converting uridine to cytidines when 
the uridine is disposed within a double-stranded nucleic acid structure at 
a position several nucleotides inward from each terminus of the 
double-stranded structure. The target uridine could be disposed as few as 
2-4 nucleotides inward from the terminus of the double-stranded structure, 
or it may be embedded within hundreds of base pairs of double-stranded 
structures. 
The RNA editing component may be comprised of one or more sub-components. 
Therefore, for purposes of the present invention, the term "RNA editing 
component" refers to either a singular component or a plurality of 
components. 
4. Double-stranded structure: refers to a complementary-base-paired 
double-stranded nucleic acid. In practicing the present invention, the 
double-stranded nucleic acid may consist of an RNA-DNA hybrid or an 
RNA-RNA hybrid. 
In accordance with the present invention, it has been discovered that 
nuclear extracts, or whole cell extracts of cells which contain a high 
percentage of nuclear material, contain a component which is capable of 
editing RNA. This editing function comprises converting uridine to 
cytidines under prescribed conditions, and was first discovered in the 
study of editing of the Hepatitis Delta Virus (HDV) RNA genome. Zheng et 
al., J. Virol., 66:4693-97 (August 1992). Examining the 
naturally-occurring phenomenon of a U to C conversion at position 1012 of 
the HDV genome during replication, it was discovered that the change from 
U to C at this position occurs independent of any viral gene products. In 
fact, it was found that extracts containing a high percentage of nuclear 
material were capable of editing the HDV genome at position 1012 in the 
same manner as observed in intact cells infected with HDV. Editing was 
observed with extracts of Drosophila embryos, HeLa cells or calf kidney 
cells. The modification of position 1012 was found to occur in the absence 
of RNA replication, and, therefore, was proven to be an editing reaction, 
rather than a misincorporation of C for U during RNA-directed RNA 
synthesis. 
It was further discovered that the entire HDV genome was not necessary for 
the modification by nuclear extracts to occur. However, the 
double-stranded rod-like structure of the HDV genome was required at the 
site of conversion, indicating that a double-stranded structure is 
necessary for the RNA editing component of the nuclear extracts to 
function. It should be noted that the modified U of the HDV genome is 
embedded in 10 consecutive base pairs of double-stranded structure. 
Following the aforementioned observations in connection with HDV, it has 
been further discovered in accordance with the present invention that even 
non-viral RNA may be specifically edited by nuclear or 
high-nucleus-containing cellular extracts. The editing is a direct 
conversion of U to C, which may be due to amination of uridine to 
cytidines. Even though any RNA sequence appears to be a potential target 
for the editing function, the target uridine must be embedded in 
double-stranded structure, such as the 10 paired bases of the HDV genome 
around position 1012, which can be achieved by hybridization with either 
DNA or RNA. 
It is likely that more than one uridine may be edited by the RNA editing 
component of nuclear extracts. However, the editable uridines must be 
flanked on either side by double-stranded structure. 
Although the agent responsible for the editing function of the 
aforementioned nuclear or cellular extracts has not been elucidated, it 
may prove to be attributable to the enzyme cytidines synthase, which 
converts UMP to CMP and UTP to CTP, both reactions being essential to all 
cells. It has been discovered that glutamine is the amino donor for both 
the cytidines synthase and the RNA editing function. Moreover, both 
reactions are inhibited by the glutamine donor inhibitor, 
deazo-oxo-norleucine. Furthermore, a line of cultured Chinese hamster lung 
cells, which lacks cytidines synthase, has also been found unable to edit 
RNA. 
Regardless of the identity of the specific factor(s) responsible for the 
RNA editing function of nuclear extracts, it is clear that nuclear 
extracts, or whole cell extracts containing a large percentage of nuclear 
material, possess the RNA editing function in sufficient quantity to 
effectively edit target RNA sequences, as will be described in greater 
detail below. 
Various embodiments of the RNA editing method of the invention are 
described below. Any molecular cloning or recombinant DNA techniques not 
specifically described are carried out by standard methods, as generally 
set forth, for example, in Sambrook et al., "DNA Cloning, a Laboratory 
Manual," Cold Spring Harbor Laboratory (1989). 
RNA molecules having a selected target sequence may be isolated from any 
biological source, such as solid tissues, body fluids, such as blood, 
lymph, urine, and the like, cultured cells, or microorganisms. Total RNA 
is isolated and purified by procedures commonly known in the art. For 
example, total nucleic acids may be isolated from cells by the use of 
sodium dodecyl sulfate (SDS) and pronase, followed by a phenol extraction 
and two ether extractions. Nucleic acids are then collected by ethanol 
precipitation, digested with a DNase (e.g., RQ DNase I, Promega Biotech) 
then extracted once more and precipitated with high-salt ethanol. A 
variety of other RNA isolation methods will be apparent to one skilled in 
the art. 
Specific RNA fractions may also be purified using known procedures. For 
example, poly(A).sup.+ RNA may be recovered from total RNA by passage 
over a poly(U) or oligo(dT) column, using procedures supplied by 
manufacturers of such column materials (e.g., Pharmacia Chemicals, Inc., 
Piscataway, N.J.). 
It should be apparent to those skilled in the art that the source of the 
RNA is selected on the basis of the RNA molecules targeted for editing. 
For example, if it is desired to modify the expression of mRNA encoding a 
liver-specific enzyme, liver tissue or cells should be selected for 
obtaining RNA. 
After RNA has been isolated and purified, it may be stored for future use 
under ethanol at -20.degree. C., for example. The RNA is then collected by 
centrifugation, dried and resuspended in a suitable biological buffer, 
such as TE buffer (10 mM Tris-HCl, 0.1 mM EDTA) just prior to use. 
Assuming knowledge of at least portions of the RNA molecule desired to be 
modified, any target sequence may be selected for editing. For example, a 
portion of an mRNA which encodes a structural feature of the gene product 
may be targeted for modification, thereby to change, enhance or interfere 
with the formation of that gene product. Alternatively, a portion of an 
mRNA encoding a signal or transit peptide, required for transport across 
biological membranes, may be targeted for modification to improve or 
disrupt such transport. Similarly, the translation start site itself may 
be modified (e.g., AUG to ACG). 
The criteria for selecting a suitable target sequence are: (1) that it 
comprises a "editable" segment having at least one uridine disposed 
therein; and (2) the editable segment is flanked on either side by 
flanking sequences several (at least 5, in a preferred embodiment) 
nucleotides in length. These criteria are important for the practice of 
the present invention, so that the target sequence may be hybridized to a 
complementary nucleotide sequence, thereby forming the requisite 
double-stranded structure necessary for activity of the RNA editing 
component of the nuclear extract. It should be appreciated that the 
flanking segments may comprise uridines; however, these uridines generally 
will not be edited by the RNA editing component of the nuclear extract. 
The single-stranded complementary nucleic acid used to form the requisite 
double-stranded structure can be either DNA or RNA. It may be prepared by 
any of several common methods known in the art. For example, complementary 
oligonucleotides may be prepared by PCR amplification of existing 
sequences. Alternatively, they may be chemically synthesized (e.g., via 
phosphoramidite chemistry) using commercially available reagents and 
apparatus. Oligonuceotides may also be modified, e.g., such that they 
possess increased stability in a cellular environment (particularly 
important for RNA), or are targeted to a particular tissue, or are more 
efficiently transported across a cell membrane. Preferably, the synthetic 
oligonucleotides should be complementary to the entire target sequence, so 
that they are capable of forming the double-stranded structure necessary 
for activity of the RNA editing component of the nuclear extracts. 
Single-stranded complementary nucleic acids may be stored lyophilized, or 
under ethanol, or dissolved in a suitable biological buffer, such as TE 
buffer. 
Suitable nuclear or cellular extracts having the RNA editing component may 
be obtained from a variety of sources. In preferred embodiments, cell 
extracts designed for use in in vitro eucaryotic transcription are 
particularly convenient and useful for practicing the present invention. 
For example, extract of Drosophila embryos supplied in the in vitro 
eucaryotic transcription kit available from Stratagene (La Jolla, Calif.) 
contains the RNA editing component(s) capable of converting U to C, 
according to the present invention. Preparation of a Drosophila embryo 
extract was disclosed by Heierman et al., Nucl. Acids Res., 13:2709-30 
(1985). Another suitable cell extract is a HeLa cell lysate, supplied with 
the eucaryotic transcription system sold by Bethesda Research Laboratories 
(Gaithersburg, Md.). Preparation of a HeLa cell lysate was disclosed by 
Manley et al., Proc. Natl. Acad. Sci. USA, 77:3855-59 (1980). 
It should be apparent to those skilled in the art how to obtain suitable 
cell extracts from other cell types. Such methods are commonly known in 
the art and will be readily apparent to practitioners in the field. 
Moreover, many of these cell extracts are commercially available. Cell 
extracts from sources other than those mentioned above may be compared 
with extracts having known RNA editing function, to determine if they also 
contain effective amounts of the RNA editing component. 
The method of the invention involves creating a double-stranded nucleic 
acid structure in the target sequence. To accomplish this, any preexisting 
secondary structure in the RNA molecule may need to be disrupted, thereby 
rendering the target sequences contained therein hybridizable. Many 
methods are available for denaturing double-stranded nucleic acid 
structures. For purposes of the present invention, it is preferable to 
denature the RNA by means which avoid chemical modification. For example, 
samples containing RNA may be heated to 65.degree.-95.degree. C. for 2-3 
minutes, then quickly cooled by placing in an ice bath. 
The denatured RNA molecule comprising the target sequence may be hybridized 
with the complementary nucleic acid according to standard methods. For 
example, RNA comprising the target sequence and the complementary nucleic 
acid may be combined in the presence of appropriate salts, heated to 
65.degree. C., then slowly cooled to room temperature, thereby effecting 
hybridization of the target sequence with the complementary nucleotide 
sequence. 
The hybrid molecule is then treated with the nuclear extract by combining 
them in a suitable biological buffer, such as 50 mM Tris-HCl, pH 7.9, 6 mM 
MgCl.sub.2, 40 mM (NH.sub.4).sub.2 SO.sub.4, 0.2 mM EDTA, 1 mM 
dithiothreitol and 15% glycerol (see Manley et al., supra), or 10 mM 
Hepes-KOH, pH 8.0, 8.5 mM MgCl.sub.2, 120 mM KCl 0.5 mM dithiothreitol and 
20% glycerol (see Heiermann et al., supra). In a preferred embodiment, 
nuclear extracts are used at concentrations equivalent to those used for 
eucaryotic in vitro transcription, according to instructions provided by 
suppliers of such in vitro transcription kits, as described above. In a 
preferred embodiment, a 25 .mu.l reaction mixture contains 100 ng of the 
hybrid molecule. The reaction mixture is incubated for up to 16 hours at 
20.degree.-37.degree. C. In a preferred embodiment, the incubation is 
performed at 30.degree. C. for 30 minutes. Reactions may be terminated by 
phenol/ether extraction of each sample, as described above, followed by 
precipitation of the RNA with ethanol. 
Editing of the target RNA sequence can be verified by DNA sequence analysis 
of the target RNA sequence, after it has been reverse-transcribed and 
amplified by polymerase chain reaction (PCR). Suitable primers may be 
constructed for PCR, according to methods well known in the art. The 
nucleotide sequence of the amplified DNA product is determined according 
to well known methods. 
In an alternative embodiment for verification of editing, if the editing 
results in the introduction or removal of a DNA restriction site, this may 
be utilized to verify the editing by a simple electrophoretic method, not 
involving DNA sequencing. In this embodiment, one of the PCR primers is 
end-labeled, thereby to form a detectable PCR product. The product is then 
digested with the appropriate restriction endonuclease, then separated by 
electrophoresis on an agarose gel. The relevant introduction or removal of 
the restriction site should readily be apparent, upon drying the gel and 
visualizing the change in mobility of the DNA segments by autoradiography. 
Thus, target sequences of selected RNA molecules may be edited in vitro 
according to the methods set forth hereinabove. The method may be utilized 
to disrupt gene expression, enhance gene expression or modify the 
structure or function of a gene product. 
The method of the invention may be modified for in vivo application, taking 
advantage of the observation, in accordance with the present invention, 
that intact cells can exert the RNA editing effect present in nuclear 
extracts, provided that the requisite double-stranded structure can be 
produced within those cells. For example, as disclosed by Zheng et al., J. 
Virol., 66: 4693-97 (August 1992), transfection of cultured cos 7 cells 
with HDV genome DNA or RNA resulted in editing of the RNA genome at 
position 1012, in the absence of any other viral functions. It should be 
noted in this connection that HDV genomes possess the requisite 
double-stranded structure at the editing position. 
Hence, the method of the invention may be modified for in vivo application 
as follows. Complementary nucleic acid sequences, as described above, are 
synthesized and prepared for in vivo delivery, according to known methods. 
Such methods include, for example, virus mediated infection of target 
cells, or delivery by target-specific liposomes or targeted cellular 
carriers, such as erythrocyte ghosts. Methods for preparing tissue- or 
cell-specific liposomes or erythrocyte carriers are well known in the art, 
as are methods for encapsulating nucleic acids therein. See, e.g., Glenn 
et al., J. Virol., 64: 3104-07 (1990). Such methods are suitable for 
targeted in vivo delivery to cells or tissues of a variety of living, 
multicellular organisms, including mammals. 
Alternatively, cultured cells may be transfected with the complementary 
nucleic acids, according to well known methods. See, e.g., Zheng et al., 
supra. The method of the invention has been performed in several cultured 
cell lines, including cos 7 monkey kidney cells, human liver cell lines 
HuH7 and HepG2, and the mouse fibroblast cell line 3T3, which are all 
widely available. 
Once the complementary nucleic acids are present in the target cells, they 
can hybridize with the selected target RNA sequence, in the same way as 
ribozymes or anti-sense RNA molecules have been shown to do in vivo. See, 
e.g., Haseloff et al., Nature, 334:585-91 (1988) and Cameron et al., Proc. 
Natl. Acad. Sci. USA, 86: 9139-43 (1989). Once hybridized, the required 
double-stranded structure in the region targeted for editing is formed, 
thereby enabling the editing component present within the cell to exert 
its effect. Thus, in a manner similar to the mode of operation of 
anti-sense RNA and ribozymes, "editing" oligonucleotides may be introduced 
into target cells to provide RNA-targeted therapy in vivo. It should be 
appreciated by those skilled in the art that the choices of target 
sequences in vivo may be somewhat more limited than for in vitro 
applications. This is because the target RNA sequence preferably should 
not be part of any inherent higher order structure, such as base-pairing 
or association with a cellular protein. 
The RNA editing methods and strategy of the present invention constitute an 
advantageous alternative to methods currently available for providing RNA 
therapy. Like existing methods (e.g., ribozymes and anti-sense molecules), 
RNA therapy may be directed to cells and tissues wherein gene expression 
is actually occurring. However, whereas current RNA therapy methods can 
only accomplish disruption of gene expression, the methods of the present 
invention provide a way to redirect gene expression. For example, an RNA 
molecule can be edited to enhance its stability or efficiency of 
translation, or to actually alter the structure or function of a gene 
product. These advantages should lead to significant advances in the field 
of gene therapy.

The following examples are provided to describe the invention in further 
detail. These examples are intended merely to illustrate and not to limit 
the invention. 
EXAMPLE 1 
In Vitro Editing of Target RNA Sequences 
The following are examples of substrates that have been tested for in vitro 
RNA editing according to the present invention. In each example, the 
target nucleotide is in bold type and underlined. 
__________________________________________________________________________ 
Natural HDV substrate, Sequence I.D. Nos. 1 (Top); 2 (Bottom) 
5'- . . . AGAGUAUAUCC .sub.-- UAUGGAAAUCC . . . -3' 
Genomic HDV RNA 
3'- . . . CUUCCCGUAGGGUACCGAGGUG . . . -5' 
Genomic HDV RNA 
Modified HDV substrate, Sequence I.D. Nos. 1 (Top); 3 (Bottom) 
5'- . . . AGAGUAUAUCC .sub.-- UAUGGAAAUCC . . . -3' 
Genomic HDV RNA 
3'- . . . UCUCAUAUAGGGUACCUUUAGG . . . -5' 
Antigenomic HDV RNA 
Modified HDV substrate, Sequence I.D. Nos. 1 (Top); 4 (Bottom) 
5'- . . . AGAGUAUAUCC .sub.-- UAUGGAAAUCC . . . -3' 
Genomic HDV RNA 
3'-GTAGGGTACC-5' Complementary RNA 10-mer 
Non-HDV substrate, Sequence I.D. Nos. 1 (Top); 5 (Bottom) 
5'- . . . AGAGUAUAUCC .sub.-- UAUGGAAAUCC . . . -3' 
Genomic HDV RNA 
3'-GTAGGATACC-5' Complementary DNA 10-mer 
Non-HDV substrate, Sequence I.D. No. Nos. 6 (Top); 7 (Bottom) 
5'-GGGAAGAGUAUAUCUC .sub.-- UAUGGAAAUCCCU-3' 
29-mer variation of HDV RNA 
3'-CCCTTCTCATATAGTGATACCTTTAGGGA-5' 
Complementary 29-mer DNA 
__________________________________________________________________________ 
In vitro editing of the above-listed RNA target sequences was accomplished 
as follows. Single-stranded target RNA sequences were hybridized with 
complementary nucleic acids according to standard methods. Extracts from 
Drosophila embryos were purchased from Stratagene. Nuclear extracts of 
HeLa cells are commercially available from Promega Biotech, Madison, 
Wisconsin. In vitro editing reactions were performed by placing, e.g., 100 
ng of the hybrid nucleic acid in, e.g., 25 .mu.l nuclear extract, and 
incubated for 30 minutes at 30.degree. C. Nucleic acids were then 
recovered by phenol extraction followed by 2 ether extractions, ethanol 
precipitation, digestion with RQ DNase I (Promega Biotech) and an 
additional extraction and ethanol procipitation. 
To verify the editing of the target nucleotide in substrates 1-4 above, the 
target sequences were reverse-transcribed to form cDNA, then amplified by 
PCR. The PCR product of Sequence I.D. No. 1 of substrate 1 was sequenced 
by a dideoxy nucleotide sequencing procedure, using one of the PCR primers 
and a Sequenase kit (U.S. Biochemical) with the products resolved on a 
sequencing gel of 6% polyacrylamide. Additionally, substrates 1-4 were 
tested for cleavage by NcoI restriction endonuclease, since editing of the 
target nucleotide introduces an coNCOI restriction site in all four target 
sequences. See Zheng et al., supra. 
Substrate 5, comprising target RNA Sequence I.D. Nos. 5 and 7 was too small 
for PCR amplification. Therefore, an alternative method for verifying RNA 
editing was employed. The RNA strand of Sequence I.D. No. 6 was labelled 
with [.alpha.-.sup.32 P]UTP, and the RNA editing reaction was carried out 
as described above. The RNA was then digested with nuclease P1 (Sigma) and 
subjected to thin-layer chromatography using Solvent 2 disclosed by Wagner 
et al., Proc. Natl. Sci. USA, 86: 2647-51 (1989) to measure the conversion 
of uridine to cytosine. Radioactivity was detected either by 
autoradiography or with a Radioanalytic Imaging System (AMBIS, San Diego, 
Calif.). 
Sequence analysis of the PCR-amplified substrates 1-4 above revealed that 
the target uridine had been converted to cytidines in each target 
sequence. The efficiency of conversion ranged from 5-60%, depending on the 
target RNA sequence and the particular nuclear extract utilized. For 
example, substrates 1 exhibited a 60% conversion of U to C when incubated 
with the Drosophila extract. It is possible that longer incubation times 
would result in even greater percentage of conversion. 
Although the thin-layer chromatography procedure does not yield information 
regarding specific editing, it revealed that the target RNA sequence of 
substrate 5 was edited by the nuclear extract in vitro, based on the 
appearance of radiolabelled cytidines during the incubation. 
EXAMPLE 2 
Editing of Target RNA Sequences in Cultured Cells 
Substrate 1 from Example 1 above was tested for the ability to be edited in 
cultured cells, according to the present invention. Transfection was 
accomplished by means of a calcium phosphate procedure, according to 
standard methods. For RNA transfections, Lipofectin (BRL) was used. Cos 7 
and related cell lines were used as host cells for the transfections. 
Following transfection, cells were maintained 3-18 days according to 
standard methods, then total nucleic acids were isolated from the 
transfected cells. This was accomplished by use of sodium dodecylsulfate 
and pronase, followed by a phenol extraction and two ether extractions. 
Samples were collected by ethanol precipitation, digested with RQ DNase I 
(Promega Biotech), then extracted once more with phenol and ethanol 
precipitated. Editing of the target RNA sequence was verified by reverse 
transcription and PCR amplification, as described in Example 1 above. 
Editing of the target RNA sequence was observed as early as 3 days (the 
earliest time examined) and increased to 18 days after transfection. At 3 
days after transfection, approximately 10-18% of the target RNA sequence 
was modified to comprise C instead of U. At 18 days after transfection, 
21-33% of the target RNA sequences was edited. 
The present invention is not limited to the embodiments specifically 
described and exemplified above, but is capable of variation and 
modification without departure from the scope of the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis delta virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGAGUAUAUCCUAUGGAAAUCC22 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis delta virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GUGGAGCCAUGGGAUGCCCUUC22 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hepatitis delta virus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GGAUUUCCAUGGGAUAUACUCU 22 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CC ATGGGATG10 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCATAGGATG10 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GGGAAGAGUAUAUCUCUAUGGAAAUCCCU29 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: YES 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AGGGATTTCCATAGTGATATACTCTTCCC29