Note: Descriptions are shown in the official language in which they were submitted.
~ACK~ROUND OF THE INVENTION
A.. Field of ~he Inven~ion
The invention generally relates to therapeutic
compositions of matter, methods for producing said compo-
; sitions, and methods for administ:ering said composition to
- living organisms, including human beings. Particularly, the
several embodiments of the invent:ion allow protection of an
organism against viral attack by stimulation of the cells of
the organism to cause said oells to produce an antiviral
protein known as interferon.
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B . Description of the Prior Ar t
Interferon is an antiviral protein released by
animal cells in response to viral infec~ion. It has long
bPen known that RNA is a specific virion component which
triggeri the release of lnterfexon in animal cells, both
natural and synthetic double-stranded RNA's belng known to
stimulate interferon production. These double-stranded
RNA molecules have not found utility as chemotherapeutic
agents due to the toxicities thereof, such -toxicity being
related primarily to the presence of the double-helical RNA
structure. It has been recently shown that the first step
in interferon induction in the cells of a liviny organism,
i.e., the absorption of the nucleic aoid complex of poly-
inosinic acid annealed to polycytidylic acid ~rI~ rCn),
is a rapid event, thereby suggesting that an intact primary
structure of the inducer complex be present only for a brief
peri~d in the cell. The present invention now concludes;that,
once interferon induction is triggered, the continued presence
of doubl~-helical RNA is unne~essary and leads to secondary
effects on the cell without increasing the magnitude of
antiviral resistance~ r~he invention thus involves provision
of douhle-stranded nucleic acid complexes which, while re~
taining the capability of inducing interferon production in
cells, are capable of being readily hydrolyzed by nucleases
in the cells. Such nucleic acid complexes are duplications of
the double-stranded virus genes which promote interferon
production during viral attack, but which are modified to
thereby be rendered less toxic to the cells due to the ability
o~ said modified complexes to be more readily destroyed within
.
--2 - : ~
the cells. The compounds of this invention have been
disclosed previously from the laboratories (J.Molecular
Biology 70:56~[1972]).
In addition, now a new type of hypoxanthine-
polynucleotides has been synthesi~ecl from a mixture of
inosine 5'~diphospha~es and 2'-~me~hy}inosine 5'-dip~osphates
with M. luteus polynucleotide phosphorylase~ The procedure
is similar to that previously disclosed f~om the laboratories
~Biochemistry 11, 4931 [1972~). This type of hypoxanthine -
polynucleo~ides contain 5-16% 2LO~methylinosine residues
together with 95-84~ inosine residues correspondingly,
designated as (rIs_20, 2'-MeI)n. These complexes, (rI5_20,
2'-MeI)n~rCn, are 100-fold more active than the rIn rCn
as inter~eron induces to human cells. Apparently this
modification of the backbone of polyinosinate strand has
;~ a pronounced enhancement effect; therefore a less amount
o~ (rIs_20,2'MeI)n-rCn is needed then rIn rCn in protectlng
the human cells against viral diseases.
SUMMARY OF THE INVENTION
. .
Interferon is normally produced by a living animal
cell on viral attack, the double-stxanded helical virus gene
triggering the production of this antiviral protein by the
cells.
Th~ action of the double-stranded viral gene can
be mimicked by the 1:1 complexes of polyriboinosinic and
polyrihocyti~ylic acids (rIn rCn). Two generally accepted
condikions for such complexes to be adequate for induction
of interferon production by cells are: (1) inkactness of
the double-stranded complex (with a Tm higher than the
,
`incubation temperature); an~, (2) adequate resistance to
nucleases, i.e., to enzymatic activi-ty in the cells. It has
presently been found that additional structural and con-
formational requirements for the (rIn~rCn) complex exist.
Particularly, interruption of th~ rCn strand in th~ complex
by unpaired bases such as uridine or guanine has less
effect on the interferon induction capability of the r~sul-
ting complex than does interruption of the rI~ strand. Thus,
for interferon induction , the structural requirements in
strand continuity and base-pairing are more stringent in
the rIn strand than in the rC~ strand. Modifications to
the rCn strand of ~he (rIn~rCn) complexes are thus possible
with retention of interferon induction capability and sig-
nificant reduction of toxicity.
The present invention provides compositions of matter
wherein duplications of these double-stranded helical ~irus
genes are modified to an extent whereby the ability to induce
interferon production in the cells is retained but the
toxicitias normally associated with such compounds are re-
duced to an extent which allows their use as chemotherapeuticagents. In the first type of modification, dot~le-stranded
nucleic acid complexes which normally induce cellular
interferon production are modified struaturally so that the
resulting molecule is readily hydrolyzed by nucleases within
the cells. The modified nucleic acid complexes retain their
ability to induce inter~eron production in the cells, but
are rendered much less toxic than the unmodified double-
stranded structures due to th~ abillty of the c~lls to hydro-
lyze the modified complexes shortly after interferon production
has been induced.
In t~le second type of modification, the ribosyl
backbone of polyinosinic acid in the rIn rCn complex is
partially replaced ~to the extent of 5-16~) by the
; 2'0-methylribosyl residues. Th~ modified complexes
(rI5_20, 2'MeI)n-rCn were found to be 100 fold more active
than rIn rCn as inducer for in~erferon in the human cells.
Toxicity reduction in the fixst type of modified
(rIn rCn) complexes is accomplished by s~xuctural modification
of said complexes, a general modification being ~he intro-
10 duction ofa nucleotide into the rCn strand to prevent normalpairing between the strands, thereby to produce a "weak point"
or chemical position on the strand which is vulnerable to
attack by nucleases in the cells. While the modiied complex ;~
retains s~ficient structural integrity to induce interferon
production in cells, this vulnerability to enzymatic activity
allows relatively rapid hydrolysis thereof withln the cells.
Thus, the modified complex structure is destroyed after its
function as an interferon inducer is served, destruction of
the complex preventing;harmful effects which would be ~aused
thereby were said complex allowed to remain in the cell. Since
the continued presence of this double-stranded, helical complex
does not increase antiviral resistance in the cell once in-
duction has been triggered, nothinc3 is lost by destruction of
thè complex,the reduction in toxicity to the cell and to the
organism allowing its use as a therapeutic agent for protection
of an organism against viral attack.
In the second type of modified (rln rCn) complexes,
the rI~ strand is replaced by a (rI5_20, 2'MeI)n~strand. This
new complex, (rI5_20, 2'MeI)n rCn~ is 100 fold more active
30 than rI~ rCn; thereore, a much lower dosage of this modifled
complex is needed for the same pxotection against viral attack
; to the human cells.
--5--
It is therefore an object o the invention to
` provide compositions of matter ancl a method for protecting
against viral infection in a cell by stimulating the pro-
duction of interferon by the cell.
It is another object of the invention to provide
a method for structuring, on a molecular scale, double~
stranded, helical nucleic acid complexes to render said ~-
; complexes capable of inducing intérferon production in an
organism while being non-toxic to the organism in therapeutic ~ -
10 dosages.
It is a further object of the invention to provide
therapeutic agents comprised of (rIn rCn) complexes which are
rapidly hydrolyable by enzymatic activity in living cells
after induction o~ interferon production therein.
It is an additional object of the invention to pro-
vide therapeutic agents comprised of (rIs_20, 2'Mel)n-rCn ;
which may be many-fold more active than rIn~rCn as human ~ ~
interferon inducer. ~;
Further objects and advantages of the invention will
20 become more readily apparent from the following detailed
d~scription of the preferred embodiment of th~ invention.
BRIEF DESCRXPTION OF THE DR~WING5
. _ _ .. . .... .
` Fig. 1 is a graphic illustration of an elution pattern~
from a Sephade~ G50 column (2.5cm x 95 cm~ of a hydrolysate
of r~C20,G~n by RNase Tl, the designations Fr. 1~6 indi-
catiny "fractions 1 through 6i';
Fig. 2 is a graphic illustratisn o melting curves
; of rI~ rCn~ r(I39~U)n rCn, and r(I2l U)n r~n in minimal Eagle's
salt solution, nucleotide concentration bein~ 4 x 10 5M in
30 1 + C;
-6-
8~9
Fic3. 3 is a yraphic illustration of melting
curves of rIn r(C22,U)~, rIn r(C13/U)n~ rIn r~C7~U)n'
rIn'r(C4 U)n, and rIn r(C20,G)n in minimal Eagle's salt
solution, nucleotide concentration bei:ng 1 x 10 M in I ~ C;
Fig. 4 in a graphic illustration of melting curves
of ~Ip)gI rCn (Nucleotide concentxation 1 x 10 4M)and
`~ (Ip)l2I-rCn pGly-L-lysine (initial nucleotide concentration
2 x 10 5M, P/N ratio - 1) in minimal Eagle's salt solution,
and (Ip)l6I rCn (nucleotide concentration 1 x 10 3MJ in
0.15m NaCl, 0.OlM MgCl~, and 0.OOlM sodium phosphate (pH7.4)j :
Fig. 5 is a yraphic illustration o~ melting curves
of (1) rIn r(Cp)4~G>P, (2) rIn r(Cp)35G~P, (3) rIn X(CP)23G~P'
and (4) rIn r(Cp)llG~p, in minimal Eagle's salt solution,
nucleotid~ concentration being 1 x 10 M in I ~ C;
Fi~. 6 is a graphic illustration of the hydrolysis by
pancrease RNase of ta) rIn rCn, (b) r(I21,U)n rCn, (c)
rIn r(C4 U~n and th~ir complexes with poly-~-lysine and
poly-D-lysine, the polynucleotide complex alone being re~
presented ~y the line X-X, its complex with poly-L-lysine ::
by the line ~ ~~ (P/N ratio = 2) and 0-0 (P/N ratio = 1),
its complex with poly-D-lysine by the line ~ (P/N ratio = 2)
and ~-0 (P/N ratio = l);
~ 'ig. 7 is a graphic illustration of the hydrolysis
by pancrease RNase of rIn'rtCp)48G?p, the polynucleotide complex ~. ;
, alone being represented by the llne X~X, its complex with poly-
L-lysine by the line 0-~ (P/N ration = 1), in minimal Eagle's
~alt solution, nucleotide concentration being 1 x 10-5M in
I + C; and,
Fig. 8 is a yraphic illustration of the hydrolysis
30 o~ r(I~n~r(Cjn, xepresented by the line ~-~ , r~I~n, repr - :
sented by the line a-o, and r(I)n'r(c20lG)n~ represented
~ -7~
8~
by the line ~-0 , with S~g/ml. of RNase Tl and pancreatic
RNase A at 37C in Eagle's medium.
DE5CRIPTION OF THE PREFERRED EMBODIMæNTS
The present compositions of matt~r and methods relative
to their use generally depend in the several embodiments there-
of on the chemical modification of an inter~eron inducing
nucleic acid complex to render said complex less toxic to a
living animal cell. The chemically modified complexes dis-
closed herein retain the capability of the unmodifiecl complexes
~or inducing the production of interfexon by cells while being
rendered more readily hydxolyzable by nucleases in the cells
relative to the unmodified complexes. The increased hydrolysis
capability o the modified complexes renders said complexes
less toxic to the cells due to the relatively rapid destruction
of the complexes after interferon induction.
The double-stranded, helical nucleic acid complexes
whlch are of concern in the present invention may be modified
by mismatching of bases, thereby causing a "looping-out" from
the helix at the point or points thereon where the bases are
caused to be unpaired; by strand-interruption formation of
oligomer-polymer complexes; and by disposition o~ hydrocarbon
grvups, such as a methyl group, within the helical structure.
In the f.irst of these particular modifications, pairing of
; bases in the two strands of the complex is interrupted to~pro-
duce a "weak" point or vulnerable chemical position in the
complex which is~subject to attack by nucleases in the cells.
Unpaired bases such as uridine or guanine interrupt either of
the strands of a nucleic acid compIex, such as the rC strand
of the complexes ~f polyriboinosinic and polyrikocyticylic acids
~poly rI-rC or rIn rCn, which is polyinosin:ic acid annealed to
--8-
polycytidylic acid)l without interferring with interferon
induction capability (~hereby preserving antiviral function).
However, this interruption permits accelerated hydrolysis of
the complex, thus reduction in toxicity thereof, because of the
formation of a weakened position in the structure of the com-
plex which is more subject to chemical attack and hereby
hydrolysis by nucleases in -the cells. Similarly, bond break- -
age in the do~le~stranded helical structure effected by
strand-interruption formation of oligomer-polymer complexes
or by disposition of hydrocarbon groups in the structure
provides sites in the molecular structure of the complex
which are more readily susceptible to hydrolyzing enzymatic
attack.
The nucleic acid complex, rIn rCn, noted above proves
particularly valuable as a basic structure for modification
into low toxicity interferon inducers. Data to be presented
hereinafter show that, for interferon induction, the struc-
~tural requirements in strand continuity and base-pairing are
more stringent in the rIn strand than in the rC~ strand.
2~ Thus, modified rIn rCn complexes wherein the rC~ strand is
interrupted prove most useful. Since it has been found that
the triggering o~ human cells for interferon production through
the absorption of rIn rCn is completed within a few minutes,
the intact primary structure of the inducer complex is required
to be present in the cell only briefly. Once the induction
has been triggered, the continued presenceof ~e double-stranded
helical rIn rCn is not necessary and leads to harmful effects
without increasing antiviral resistance. Accordin~ to this
kinetic factor, modi~ication of the rIn rCn complex to a more
`` `
readily hydrolyzable form as previously described does not
reduce antiviral resistance. Two modified complexes of
rIn-rCn which have been found to retain the interferon in-
duction capability of the unmodiied complex but which are
re~atively rapidly hydrolyzed by nucleases within the cells
n ( 12-13'U)n and rIn r(~20_29~G)n where U represents
uridine and G represents guanine. Other complexes so struc-
tured include rIn-r(C22,U)n and rIn r(C7, )n
of and characteristics of these modifi~d complexes will be
described hereinafter.
Exposure of an organism to massive dosages of either
the unmodified rIn rCn or the modified complexes results in
toxic ~ffects due to the inability of the organism to effective-
ly dispose of~said substances within a sufficiently short
period of time. ~owever, those dosages normally associated
with therapeutic prac~ice are rendered harmless with the
modified complexes through enzymatic hydrolysis in the cells
much more readily than in the unmodified rIn rCn. Modif-ication
- of the backbone of the nucleic acid complexes can even produce
greater induction capability as is evidenced by the (rI6_19,mI)n-Cn
complex which exhibits 100-fold moxe activity than rIn rCn
as an interferon inducer in human cells. Effective dosages of
these modi~ied complexes vary depending on the interferon in-
duction capability and hydrolysis rates thereof, a range of
- from 1 to 100 ~ gm/kilogram of body weight being safely and
effectively administered, for example,to mice.~ Such con-
centration of the unmodified rIn rCn complex are highly toxic.
Interruption of the acid strands in these-nucleic
acid complexes produce imperfect, ox modified, complexes
having Tm values substantially higher than 37C~ the modified
complexes being protectable from nucleases by complex formation
--10--
f~
with polylysine without im~airment of the induction
ability thereof. Addition of poly L-(or D-) lysine
to rI~I oligocytidylate complexes, e.g., rIn~(Cp)23G~p,
or to the modified complexes specificaLly pointed out
hereinabove rendered said complexes resistant to enzymatic
destruction without a~fecting antiviraL activity.
The followiny m thods, procedures, and materials
have been used to conduct portions o the work resulting
in the present invention, a discussion of which is provided
for a better understanding of the practice of the invention.
Source, Composition, and Preparation of Substances
Used
Enæymes and nucleoside diphosphates: Polynucleotide
phosphorylase ~Micrococcus luteus, lyophilized powder) was
purchased from PL Biochemicals, Inc., Milwaukee, Wisconsin.
Bovine pancreases RNase, RNase Tl and Escherichia coli
alkaline phosphatase were obtained from Worthington Bio-
chemical Co., Freehold, New Jersey, IDP (trisodium salt)~
and UDP ~trisodium salt) were obtained from Miles Laboratories,
ElXhart, Indiana. CDP (trisodium salt), CDP (trilithium salt)
and GDP (disodium salt) w~re purchased from PL Biochemicals,
Inc., Schwarz BioResearch, Inc., Orangeburg, New York, and
from Calbiochem, Los Angeles, California, respectively.
The preparation of 2'-O-Methylinosine 5'-diphosphates
or related 2'-O-alkyl nucleotides have been recently disclosed
in a paper cited in Biochemistry, ~1972) 11, 4931 entitled
"A Novel Procedure For the Synthesis of 2'-O-Alkyl Nucleotides",
by I Tazawa et al.
Polynucleotides
rIn and rCn were purchased from Miles Laboratories,
Elkhart, Indiana. The maximum molar extinCtion coefficients
--11--
of 10,100 (in 0 005~ sodium acetate, pH 6 0) and 6300
(in 0-01 M-sodium phosphate, pH 7~5l were used for rIn and
rCn, respectively. r(I3g,U)n, r~I21lU)nt r~C22lu)n~
r~C4,U)n and r(C~0 G)n were prepared in the laboratory by
enzymic polymerization of nucleoside diphosphates using
polynucleotide phosphoryIase. The reaction mixture con-
tained nu~leoside diphospha~es t40 mM), 0 15 M-Tris HCl~pH 8~2),
10 mM-MgCl2, 0~4 mM-EDTA and M.luteus polynucleotide phos-
phorylase ~2 mg/ml. of the reaction mixture). After incubation
at 37 for 5 to 7 hr, ~ l vol. of 5% sodium dodecyl sulfate
and 0-1 vol~ of 10% phenol were added to ~he r~action mix- -
ture, which was shaken for 5 min. Crystalline phenol
(approximately 1 g/Sml. of the mixture) was added to the mixture,
which was shaken viyorously. The aqueous layer was separated by
contrifugation, transferred to another container, and was
treated with phenol once more. The final aqueous layer was
collected and dialyzed successively against 50 ~M-NaC1/5
mM-EDTA, 5 mM~NaCl/0.5 mM-EDTA and distilled water. AEter
dialysis, the polymer solution was free from the nucleoslde
diphosphates, as determinad by paper chromatography. The
polynucleotides in aqueous solution were stored at -17C.
Base compositions o the co-polymers were determined by hydro-
lysis of the polymer to its constituent nucleotides, followed
by conversion to the corresponding nucleosides by E. coli
alkaline phosphatase~ The nucleosides were separated by paper
chromatography and quantitated by u.v. absorption. The
polymers were hydrolyzed by 0 3 M-KOH except for r(C20,G)n
which was hydrolyzed by a mixture of pancreatic I~ase and
RNase Tl. The following solvent systems were used for paper
chromatography; isopropanol/water, 7~3 for copoly(I,U)'s;
-12-
1 n-butanol/Eormaldehyde/wa-ter~ 77 : 10 : 13 for co-poly
(C,U)'s; isopropanol/ammonia/water, 7 : 1 : 2 for r(C20,G)
The molar ex-tinction coefEicien-ts of r(I3g~U) and
r(I21,U)n ~ere assumed to be the same as that of rI .
Likewise, the rCn value was used for r(C22,u)n, r(C13,U)
and r(C20 G) . E~tinction coe~icients of r~C7,U) and
r(C4,U) have been determined to be 6700 and 6800 (in 0 01 -
M-Tris HCl, pH 7 5), respectively, by phosphorus analysis.
Table 1 summarizes the preparation, base composition and
sedimentation coefficlent of the copolymers prepared.
~.
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o~q ~
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tn
o a~
h L. r-l r-l ~1 ~ T-l r-l ~1
O ~1 ~ ) 1` 0 r-; ~ L, a)
~ C~ O~n~ ~) o ~ . ` ~ ~.
V O I~ 11 11 11 1111 11 r-l
D D P ~ P V
L~ O td O ~ r-J r~ )
rl ~ (d S I
¢l r >~ : u~ u~
~ ~a) ~ ,
C~ ~ ô~ ~ 0 03 ~ o ~ s- F~
~ A-- ~ ~ ~ ~ F ~ ~ h
~ ~1 .,~
~ ~0) ~ ~ ô o ~ r~
r~ c,~ ~ ) o o o o ~o o ~ ~:
~ O o ~ ~
h F~3 ~ _ ~ _ o
P~ ~ a a a a aa a ~d r~
u~ c) P P P P 3 P C~ ,a) ,~
.~ + + + + -
~I N O O 03 co O O O
. ~ ~ O O 1` ~` O O o _~
.~ ~ ~ L~ _ _ _ , ~)
~ u~rl ~ ~a c) ca)~ a ca a ~ ~
~ rd, - :
- ~ D D P~ ^ C!~ u~ H
~1 cn r-l N ~ ` ` O t~l --
a~ ~ t~ 1 ~ ~ ~ Q)
~ H H t.) C) C.) ,) _, r~ :
., O r~ r~ r~ r~ r~ ~ r~ * 0~
O O O O O O O ~H
; ~ O
': .
:
The preparation of the poly 2'-O-methylinosinic
i5 al50 similar to that disclosed in Biochemistry ~1972) 11,
4931, entitled "A Novel rocedure For The Synthesis of
2'~0-alkyl nucleotides", by I Tazawa. However~ in this
procedure, an attempt has been made to make a hybrid con-
taining ~oth residues, i.e., the residues of inosinic as
well as 2'-O-methylinosinic, both r2sidues being synthesised
together in the same strand in the new type of polynucleotides.
In this case, a certain proportion of both substrates are
put into the mixture, i.e., certain proportions of inosinic
5'-diphosphate and certain proportions of 2'-O-methylinosinic
5'-diphosphate were put into the enzyme reaction mixture.
Incubation followed quite closely as that reported in
Biochernistry~1972) 11, 4931, entitled "A Novel Procedure For
The Synthesis of 2'-C-alkyl nucliotides", by I. Tazawa, for
the production of poly Z'-O-methylinosinic and as quoted
in this embodiment for the synthesis of pGly 2'-0-inosinic.
Oligoinosinates
Oligoinosinates were prepared by controlled alkaline
hydrolysis of rIn, followed by treatment with HCl and alXaline
phosphatase. They were isolated by DEAE-cellulose column
chromatography and were characterized. The following
extinction coefficients were used for the oligomers: 11,400
for ~Ip)2I, 10,800 for (Ip)5I, 10,400 for (Ip)gI, and 10,200
for ~Ip)l6I.
Degradation of r(C20,G)n with RNase T
About 600 optical density units (at 268nm) of
r~C20,G)n were incubated with 350 units o RNase Tl at 37C
: for 3 hr in 12 5 ml. of 0 05 M-Tris EICl (pH 7-5)/lmM-EDTA.
Af-ter incuba-tion, the reaction mixture was lyophilizedr
dissolved in 1 ml. of water and applied to a column of
SephadexR G50 (2-5 cm x 90 cm). The column was eluted
with water, the elution profile being shown in Figure 1.
Six fractions were arbitrarily selected, as shown in
the Figure. Fractions 5 and 6 had similar u.v. spectra
to cytidine and guanosine, respectively and were not
characterized further. ~ractions 1 to 4 all showed u.v. spectra
similar to that of poly C(~m~x268 nm in 0 01 M-Tris HCL, pH
7 5). The general formula of (Cp) G~p was assigned to these
four fractions, considering the specificity and the amount of
RNase, Tl usad. Incubation of these fractions with a mixture
of pancreatic RNase, RNase Tl and E. coli alkaline phosphatase
gave cytidine and guanosine exclusively. The average chain-
length o~ these fractions was determinecl from the cytidine :
guanosine ratio, and is shown in Table 2. The following
, . . .
extinction coefficients were arbitrarily chosen for fractions
, , :
1 to 4, considering &'s of oligocytidylates and the presence -
of one guanylate residue pex molecule: fraction 1, 6600;
fraction 2, 6800; fraction 3, 7000; fraction 4, 7?00, in
0- 1 M-Tris HC1, p~ 7 5, at room temperature.
: . '
.,
-15-
:
; ~
t ~
'
_~BLE 2
~verage Chain Length and Base Composition o~
the various Fractions of (CpjnG~p obtained by
RNase T1 hydrolysis o~ poly (C20,G)*
. . .: ,,
F`rac-tion Cytidine Guanosine C:G ratio A~erage
(nmoles)(nmoles) chain length
_
,
1 2212 47 2 ~6-9:1 ~9 1
1753 35-6 ~9-2:1 ;
2 1405 38-~ 36 3:1 36~1
, 1559 46 5 33-5:1
1399 62-1 22-5:1 24-1
: 1312 55~7 23~6:1 ~
1001 86~5 11-6:1 :
4 979 86~6 11-3:1 12-4
__
:,
*Experiments were carried out in duplicate. ~-
. ~
:
,:
' ' ~:
` : -
-15~-
! ~B;
B~9
Formation of poly-and/or oliqonucleotide complexes
rIn was mixed with rCn, co-poly(C,U) 15, r(C20,G)n and
(Cp)nG~p at room tempe~ature in buffer A to form the
co~plexes; likewise, rCn was mixed with co~poly(I,U)'s and
oligo(I)'s. Since (Ip)2I and (Ip)5I do not form complexes
with rCn at room temperature, their mixtures with rCn were
kept at about 4C before experiments. All mixtures were
kept at least 3 hr at appropriate temperatures to ensure
complete complex formation before experiments. Stoichiometry
of the mixing was always 1 : 1, with respect to inosine and
cytidine residues and concentrations of the complexes were
. ~ expressed in terms of the I C residues.
Formation of the complexes between polylysines and
the poly-andJor oligonucleotide complexes
Pxeformed polynucleotide complexes in buffer A were
added dropwise to ~n equal volume of the polylysine solutions
in the same media, with stirring. Polynucleotide and
polylysine concentrations wexe adjusted to give the desired ~ ~;
value upon mixing equal volumes of the solutions. At the
concentration level of 1 to 5 x 10 5 M-residue of poly-
.
nucleotide, formation of 1 : 1 complex with poly-L-lysine
or poly-D-lysine at room temperature does not lead to a
significant amount of precipitation. This was indicated by the
~bsence of tuxbidity ~elevation of absorbance at 320 nm) and
little loss (less than 10%) of material after centrifugation
at 3000 g or 15 min. However, such a solution always
tuxns cloudy and begins to absorb strongly at 320nm, during
the melting process, at elevated temperature, and upon
cooling. It was concluded that the complex between single-
30 stranded polynucleotide and polylysine is even less soluble
:
~ -16-
than the complex of polylysine/r (I) n- r (C) n~
For the pxeparation o.f r(Ip)l2I.rCn~poly-L-lYSir1e
t~rnary complex, r(Ip~l2I rCn was made in 2 m-NaCl/O Ol
~q-NaPO4/0~ 001 M-Mgcl2 and was kept at about 5C for 1 hr.
Thsi solution was added to the cooled poly-L-lysine solution
in the same solvent. This solution was c1ialyzed at 4C
successibely against 0 Ol M-NaPO4/0~ OOl M-MgCl2 containing
1 M NaCl (for 4 hr), 0 5 M-MaCl ~for 4 hr), 0 25 M NaCl (for
10 br) and i~11y 0~15 M-NaC1 (fo~ 6 hr).
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8~91
1 ~Iydrolysis of Nucleic ~cid Complexes and the
Ternary Complexes with Polylysines by Pancreas
RNase
rIn r~nr r(I21~U)n~rCn, rIn r(C4,U)n, rIn (CP)48
and their complexas wi-th I- and D-polylysine were prepared
as mentioned above. The nucleotide concen-tration used
was lx10-5~, and the ratio of phospha-te (nucleotide) to
nitrogen ~lysine)was 1 : 1. The low nucleotide concentration
was employed to avoid precipitation of the complex by
polylysine. Bovine pancreas RNase was dissolved in
distilled water to a concen~ration of 1 mg/ml.; 60 ~
of the enzyme solution was then added to 36 ml. of the complex
solution and mixed well. The mixture was kept at room tem-
perature; its absorption spec-tra was recorded at appropriate
time intervals using a cell with a 10-cm path-length.
Experiments have been done to ensure that the lack
of increase of absorbance of the polynucleotide complexes
; with polylysine in the presence of the nuclease is a reliable
indicator that the polynucleotides remain intact. This was done
by addition of saturated (NH4)2SO4 solution to a final
concentration of 20% saturation. Under this condition, the
nuclease activity is prohibited and the polylysine-poly-
nucleotide complex dissociates. No increase in absorbance
of the polylysine-po~nucleotide complex (1:1) after incubation
with the enZyme due to the addition of (NH4)2SO4 was found
(after adjustment for dilution). Addition of (NH4)2SO4
;~ did not change the increase of absorbance caused by poly-
nucleotide degradation after incubation with enzyme in the
absence of polylysine.
~ 30
~.
Physico-Chemical Properties
~ bsorp-tion spectra were measured in a well-known
fashion.
The Tm was defined as the temperature at which
half of the total op-tical change occurred. Sedimenta-tion
coefficients were determined on a Spinco model E analytical
ultracentrifuge e~uipped with a photoelec-tric scanner.
The concentration of these samples was adjusted to give
0-5 to 0 8 optical density unit a-t 265 nm.
Biological Studies
Solutions
Buf~er A is 0 15 M NaCl, 0 01 M-sodium phosphate
(pH 7 2), 0 001 M-MgC12. This buffer was used for both
physical and biological studies so that the results could
be compared directly. Minimal Eagle medlum was prepared
to contain either 12% or 6~ ~etal cal~ serum as specified,
and glutamine (2mM), penicillin G (200 units/ml.? and
streptomycin (200 ~g/ml/) wera aaded just before use. Saline -
D is 0 14 M-NaCl, 0.0017 M.Na2~PO4, 0-0054 M RCl, 0-0023
M KH2PO~ and 0-6mg of phenol red per ml. Trypsin ~0 2%)
was prepared in saline D to contain 0 005 M.ethylenediamine-
tetra acetic acid. National Cancer Institute medium 2X
was used as the plaque-overlay medium.
; Cells
~luman neonatal fibroblasts, grown as monolayers, were
maintained in 75 cm2 plastic flasks; for interferon studies,
they were transferred to plastic panels (35 mm x 10 mm).
These cells, which produce relatively small amounts of
extracellular interferon in vitro were harvested and passed
in the standard manner.
~ .
-19A-
., . ~ .
8f~
1 Preparation of virus stocks
Bovine vesicular stomat.itis virus, New Jersey serot~pe,
was harvested Erom in:Eected mouse embryo and mouse amnion
cells to yield a titer of 1 -to lOxlO~ plaque-formin~ units/ ~-.
ml.; the virus stock, ususally diluted 100-Eold, was stored
at -70C,
'~
, ' -
'~ ' ' .' '
-19B-
.
.
1 Interferon Induc-tion a~cl ~ssa~
To ob-ta:in a sensitive measure oE interference,
several measurements of the antiviral state were quanti-
tated simul-taneously; ~a) interferon assays and two in-
dices oE intracellular interference; (b) colorimetric
assay of viral cy-topathicity and (c) reduction in viral
yield. The latter method enables us to compare the an-ti-
viral protection in the regions near 0% and 100% of cell
survival, where the former method fails. Usually the cells `
were used between passages 7 and 25, because the sensitivity
of these cells to rIn rCn changes from time to time, each
set of experiments contains an internal reference, usually
rIn-rCn studied at 10-3 to 10-5M.
Cells were e~posed to polynucleotide complexes in
minimal Eagle medium at concentrations specified for 1 hr
and then washed three times before reincubation in fresh
medium at 37C. (a) Interferon was harvested from the
e~tracellular fluids 18 hr later and measured colorimetri-
cally using bovine vesicular stomatitis virus as ~he ~
challenge virus. Assays were carried out in duplicate or
txiplicate and reference interferons (supplied by the
Biologic Resources Branch, National Institutes of Health)
were also processed from time to time to confirm the sen-
sitivity of the assay system. Assays of intracellular
interference: (b) Intracellular resistance as determined
colorimetrically. Generally a multiplicity of infection
of approximately 1 plaque-forming unit/cell was usea.
Resistance (~)was defined as the ratio of number ~f viable
c~lls (after virus infection), to the number of living cells~
(only mock infected). The colorimetric titration (in
duplicate) was usually made 72 hr after infection.
,:~
-20-
'.~
.~ . .
1 (c) Reduction in virus yield as a measurement o-f intracellular
interference. Cells were treated with bovine vesicular
sto~atitis virus (107 plaque-forming units per dish, multipli-
ci-ty of in~ection approxima~ely 20) for 60 min and the unabsorbed
virus then removed; the cells were washecl twice and the cultures
incubated for 20 hr in fresh medium. Virus titer was then
determined (in duplicate~ in mouse cells by plaque titrations.
Criteria for Idenki~ication o~ Human Interferon
~he interferons were shown initially to fulfill these ~ `
criteria: they were trypsin-sensitive, non-dialysable,~and did not
sediment at 105,000g for 2 hr. They demonstrated species speci- ~`
ficity, as shown by the absence o~ activi-ty in mouse or chick
cells. Representative interEerons obtained in ~hese studies
were chromatographed on SephadexR G150 and G200, yielding a single
peak of activity corresponding to a molecular weight of approxi-
mately 96,000 daltons.
Interpretation of Results
The General Approach
It has been generally recognized that in order for the
ribosyl polynucleotides to be e~fective interferon inducers,
they must possess the secondary structure of a double-stranded
helix. In addition, polynucleotides need to be resistant to
; nucleases in order to remain as macromolecules for a
'
~ .
.
-20A-
,
8q~
1 sufEicient lencJth of -time; polynucleo-tides are usually
much less sensitive to att~ch when -they are in a helical
complex. Therefore, -these two basic requirements have to
be recognized for an~ modification of the rIn-rCn complex.
For instance, the modified rIn-rCn complex (in
Eagle's balanced salt solution, buffer A) should have .~-
a Tm substantially above the incuba~ion t:emperature (37C)
of the cells. The sensi~ivities of these modified complexes
to nucleases have been assayed; when these compounds have
been found to be more susceptible than the parent rIn-rC
complex, measures were taken to increase their resistance.
The remedial measure adopted for such a purpose is the
introduction of polylysine (both the D-form and the L-form~
for the formation of polycation-~Olynuclqotide complexes,
which have been shown to be very resis-tant to nucleases~
Thi~ leads to an investiga-tion of the biological activity
of the polylysine-polynucleotide compléx. It is important
to show that the addition of the polylysine to rIn-rCn does
not significantly alter the interferon-inducing capability ~ :~
of the original polynucleotide complex. With the above
-strategy in mind, we have exc~mined the modified rIn-rCn
. complex with respect to its thermal melting properties,
its biological activity as a~ interferoll inducer in human
cells, its sensitivities to nucleases and the effect~oE
. -compléx formation with polylysine on all these aspects. ~:
- rI rCn Complexes containing unpaired
n Bases
~- Uridylate residues were introduced into the rIn strand and
the rCn str~d as the unpaired bases andin one special case
guanylate was introduced to the xCn strand ~or the same :
; 30 purpose. The composition of these pres~mably random co-
,~
~ -21-
~ .
.
.
.
8~3
1 polymers and their sec1imentati.on coe~icien-ts are shown
in Table 1. These polynucleo-tides have a molecular
weight ranging from 30,000 to 100,000 as judged from their
sedimentation coefficient values~ The melting profiles and
the Tm values of seven imperfect I~C complexes in bu:Efer A
(Eagle' 5 salt solution), along with the parent rIn~rCn are
shown in Figures ~ and 3. All of the complexes gave highly
co-operative profiles with the Tm ~50 to 60C) sli~htly lower
than that of rIn rCn(64 8C)/ bu-t substantially above 37C,
the incubation tempera-ture. The small reduction in Tm and
slight broadening of the helical-coil transition profile
are to be expected from -these imperfect complexes.
~; : ',' '
-
;'' ," ''
: '
, _
;
.
-21A-
'
1 When these imperfect complexes were evaluated for
antiviral func~ions (Table 3~, it was immediately apparent
that the introduction of U into rIrl strand caused a much
greater reduction in ac-tivity of the resultant complex than
when introduced into the rCn strand. The biological acti-
vities of rIn-r(C13,U)n and rI~ r(C7,U)n are significantly
higher than those of r(I3g,U)n rCn and r(I21,U)n rCn in two
separa-te experiments (Table 3), even though all of these
complexes are helical at 37C. The Tm of r~I39,U)n rCn
was only 2 deg.C lower than that of the rIn~rC~ (Fig.2),
but the biological activity is reduced by about 100-fold.
On the other hand, while the rIn~r(C13,u)n ha~ a Im of about
5 deg~C lower than that of rIn-rCn, the biological ac-~ivity
of this imperfect complex is only slightly xeduced. Even
the rIn r(C7,U)n is definitely active, although the activity
o~ rIn-r(C~,U)n is just marginal and is not resE~onsive to
an incrëasè in concentration. Previously, lt was noted
that formation of an r(C,U)n co-pol~mer with a 1:1 ratio
results in a biologically inert rInor(C,U)n complex.
Within experimental error, rIn r(C~2,U) is jUSt as active
;~ as rIn r(C)n
Oligo I-rCn and rIn-oligo C complexes,
the E~fect of S-trand interruption
It has been shown that the biological activities o-f
poly (C)-hexainosinate complex were only negligib:Le without
the enhancing effect of DEAE-dextran.
In the present studies, we examined this system in
more detail and tried to establish the requirement of chain~
lenyth of eithex oligo(I~ or oligo (C), w~ich, when complexed
with the complementary polymer, would xetain the interferon--
inducing ability of the poly (I,C) complex. The thermodynamic
~nd optical properties of oligo ~I) poly~C) comp:Lexes have
22-
~i
. .
'
1 been described in detail.
The Tm o:E the oligomer-polymer complex is dependent
on -the o].igomer chain-length and on the concentration oE the
complexes and ls less than that of the polymer-polymex
complex. The melting profiles of r(Ip)9I rCn (assayed by
absorbance) and of r(Ip)l6I rCn ~assayed by optical rotation)
are shown in Flgure 4, and some of the Tm values of the
complexes are listed in Table 4. The complexes of r(I)n~o
rCn have Tm values lower -than 37~C; thereforer the lack of
biological activity of these complexes (Table 4) is no-t
surprising. In the case of rCn r(Ip)l6I, the incubation
temperatuxe during the exposure ~1 hr) was reduced to 30C instead of 37C
~a practice which has been found in our laboratory to have
no significant effect so that the oligo I rCn complex
(Tm ~ 47C) could remain helical during the exposure. Under
this condition, the biological activity o-f this oligo I.rCn
complex was found to be only marginal, even at a concent-
ration of 10 3M. For ther(Ip)l8I~rCn complex (Tm~y50C),
definite indication of biological activity was observed :~
(Table 3), even though it is at least 100-fold less than -~
rIn-rCn.
-22A-
. .
h ~ o o o I I I I ~ r~ I o
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1 The interruption of the rCn strand was carxied out
by co-polymerization of G~ into rCn, followed by the
RNase Tl en2yme degradation. The G residues serve both as
vulnerable positions for specific hydrolysis and as identification
of chain-length, since the end residues in all khe oligomers
are the G residue. Therefore, the rIn~r(Cp)I~p complexes not
only have a strand interruption but also a looping-out of the
G residue at every interruption. The melting profiles of all
the rIn~r(Cp)IG>p complexes are shown in Figure 5. The
rIn~oligo C complexes do have
~!
':
' ~
~; 30 ~ ~
,,
. . ,
~ 23A-
-
'` ~ ' ~, ' ': " . .
lo~er Tm values -th~n the rCn oligo 1 with oligomer oE the
sa~e chaitl length. There~ore, in terms of thermal stability,
rIII r(Cp~3,G~p is approximately equivalen-t to r(Ip)~
rCn under similar conditions. The biological activities
of these rIn-r(Cp)IG~p complexes are lower than the original
rIn r(C20,G)n complex, which is almost the same as the
rIn rCn. The complex with the largest o:ligomer, rIn r(Cp)48
G ~p, has a very high T (59 6C), but is tenfold less active
than rIn rtc2o~G)~ rIn r(~ )23G~p is about tenfold more
active than r(Ip)l8I-rCn. r~n r(Cp)llG~ p showed only a
small amount of activity, as may be antlcipated, since the
Tm of this complex is below the 37~C incubation temperature.
Two conclusions can be drawn from the above results.
First, interruption o one of the strands in the helical
complex definitely reduced the interferon-inducing activity,
as exemplified by the comparison between rIn-r(C20,G)n versus
rIn r(Cp)4~G~p (Table 5). In this case, the rIn r(Cp)4g
G> p has almost the same Tm (59-6C) as that of rIn-r(C20,G)n
(60 9C) and the rI~ r(Cp)48G> p contains even less frequency
of unpaired G; yet the biological activity of rIn r(Cp)~8 ;~
G~p is -tenfold less than that of the uninterrupted
rIn r(C20,G)n. Second, the rIn oligo C complex is more
biologically active than the oligo I-rCn complex, even when
they are similar in thermal stability and not greatly
dif~erent in oligomer chain length. Thls is illustratèd in
the comparison between rIn r(Cp)23G~p versus r(Ip)l8~I rCn.
in which the former conplex is tenfold more active than the
latter. This observation reinforces the conclusion that
modification on the rIn strand has a larger biological
e~fect that that on the xCn strand.
-24
)q
:
.
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h ~ ~_
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h $ ~ h ~I S-i h h S i 5-1 hi h S-i h h h h h r~
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; . . ' .
.~ , ' ' ' . ~' ~, . . .
:
1 The above modifica-tions introduced to -the rIn rCn
complex (mismatching and interruption) invaria~ly led to a
loss of biological activity in varying degrees. Before
this observation can be interpreted properly, we should be
certain that this loss of biological activity is not simply
a reflection oE the increase in sensitivity of these ~odified
complex~s to attack of nucleases. As indicated in Figures
6 and 7, the biologically inactive r(I21,u) rcnand rIn r~C4,U)n,
as well as the less active rIn~r(cp)48G ~p, are considerably
more sensitive to attac~ of pancreatic RNase A than the highly .
active rIn~ rCn .
:
~ ' ~
~ '
~ .
:' ,
,
~ -25A-
.. l.. ~ '
., .
One ~nown procedure to protect RNA from nucleases
is by complex formation with a polycation. Interaction o~
poly-L-lysine with rIn rCn was first investigated by Tsuboi,
Matsuo & Tslo (1966); they reported a stoichiometry of I
lysine: 2 nucleotides in a solution of dilute sal-t (0 05~q-
NaCl, 0 001 M-sodium citrate/ p~f 7 0). ~his stoichiometry
was reflec-ted by the proportionality of a two-step melting
profile, lower transition (57C) for the uncomplexed rIn rCn
and a higher transition (^~90C) for the ternary poly-L-lysine
+ rIn rCn complex.
It has been shown that the binding of poly-L-lysine
to RNA(lmg/ml.) on an equivalent basis resulted in the -
forma-tion of insoluble complexes at low sal~ concen-tration.
Soluble complexes are formed, howevex, at a low poly~L-
lysine/RNA ratio. Digestion of the soluble complexes by
.
pancreatic ribonuclease, ribonuclease T1, non-specific
ribonuclease from Bacillus cereus, and Micrococcal nuclease
- yielded a precipitate wi-th a lysine/nucleotide ratio of `
l:l Together with other supporting expeximents, it was
concluded that the complex formation between RNA and poly- ~;
L-lysine protected the RN~ from attac~ by these nucleases.
Our recent reinvestigation of this interaction showed that,
in Eagle's salt solution (0-15M-NaCl, 0-00l M-MgC12 and
0-01 ~-PO4,pH7.2), the stoichiometry of the ternary complex
of poly-L-lysine to rIn rCn is l lysine: l nucleotide,
instead of 1 : 2 observed earlier in dilute salt.
A two-step transition profile was observed in
~i solu-tions with poly-L-lysine present in less than 1 : l
stoichiometry. The Tm of the ternary complex is a~out
30 83 ~ 1 deg.C. At a low concentration of rIn-rCn(less than
, 5x10~5M), by adding an equal volume of nucleic acid solution
,' ' '
-26-
., i . .,
: ' '
34~ ~
l slowly with mixing to a poly-L-lysine solution at room
temperature, precipi-ta-tion can be kept to a minimum ~less
than 10%). EIowever, such a solution invariably became
cloudy at elevated temperature, especially near the Tm~
This phenomenon, the precipitation of the melted complex
in the presence of poly-1-lysine, was veri-fied by the
observation that the single-stranded rIn and rCn are much
; less soluble than the double-stranded complex (rIn rCn)
in the presence of poly-L-lysine. We also have investi-
gated the formation of rIn rCn with poly-D-lysine. Many
; properties of this ~ernary complex are similar to those ~:
of poly-L-lysine, except that the Tm f this ternary com- ~; ;
plex is only about 68C.
As shown in Figrues 6 and 7, in the ternary complex
~ with either poly-L-lysine or poly-D-lysine in a l:l ratio
(N/P), the modified or unmodified rIn.rCn complex was
mostly protected from the pancreatic RNase. Interestingly,
in the complex with a l lysine : 2 nucleotide ratio, merely
;. half of the polynucleotide complexes were protected. This
~; 20 observation confirmed -the l:l stoichiometry of these ternary ~ .. ;
~ complexes.
: Table 6 shows that~oly-L-lysine alone does not prevent
the multiplication of bovine vesicular stomatitis virus.
Ternary complex of rIn~rC~ with poly-L-lysine iA
: ~ ' ' :'
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1 1:0-5 (P/N) ra-tio, 1:1 ratio, and 1:5 ratio, all have about
the same biologica:L activi-ty as the uncomplexed rIn rCn.
Complex formation wi.th poly L lysln~, however, does not
significantly enhance the ac~ivity of rIn.rCn either,
when rIn.rCn was supplied in suboptimal concentra-tion
(lxlO 6M~ Table 7).
Results given in Tables 4, 5 and 7 show that in
the ternary complex formation with either poly I, lysine or
poly D lysine, in 1:1 ratio or 2:1 ratio (N/P), the
~o illterferon-inducing activity of the modi~ied rIn-rCn complexes
has not been increased. For example, though the
susceptibility to nucleases of r(I21,U)~.rCn and rIn.r(C4,U)n
are greatly reduced by complex formation with poly L lysine
(Fig. 5`, the biological activities of these inactiVe, modified
complexes were not enhanced (Table 7). Similarly,
'
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1 the activities of the moderately active rIn rtcp)35G~p and
rIn-r(Cp)23G>p were no-t promoted by fcrmation of a ~:1
: complex with poly^L lysine (Table S)~ even though this
tern-ary complex should have reduced the susceptibility oE
the polynucleotides to nucleases, as extrapolated from the
results on rIn r(Cp)48G (~ig. 7). In addition, the
Tm o the rCn-r(Ip)l2I has been increased to 45C~
significantly above the i.ncuba-tion temperature, by complex
formation with poly L lysine, and this rCn oligo I complex
remained inactive (Table 4). Therefore, the reduction of
susceptibility to nucleases and the enhancement in Tm
brought about by the ternary complex with polylysine
(~ or D) did no-t transEorm the inactive, modified
rIn-rCn comp:exes into an active state.
- , ',~
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~: 30
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~ 28A- ~
1 Rela-tive Rates o Enzymic Hydrolys_s of Several
C plexes With Similar Inter~eron Induc ng
Ac-tivities
:~
As described in Tables 3 and 5, xIn r(C13,U) and
~In.r(C~O~G) are essen-tially as active in interferon
induction as unmodified rI rC ~ It is of both
theoretical and practical interest to compare the
susceptibility of these complexes toward nucleases. The
Tl ribonuclease, an endonuclease for the rI strand and G
10 residue, and the pancreatic RNase A, an endonuclease for the .
rCn strand, were both used .in this experiment. . .~.
The data in Figure 8 clearly show that both :.
.
. modified complexes were hydrolyzed at rates o~ 5~to 8-fold ~ .
. . , ~
; more rapidly than rIn rCn; indeed, approximately 50- of
their total hydrolysis occurred within the first 12 minutes ~ .
of incubation. Previous work has suggested that high - ~.. ~.`.-
antiviral activity of synthetic polynucleotide inducers . :~ :
is integrally associated with their prolonged stability .
in biological fluids; and, as greater antiviral activities
20 are achieved further toxicities simultaneously acarue. .
It has been ~ound that it is possible to prepare
:~ a polynucleotide complex which is highly active as an - ::
interferon inducer but also highly susceptible to nucleases.
. .
Two types of structural modifications were made ~ :~
~ : :
to the rIn rCn ccmplex in this investigaticn~
Mis~!atching of kases, which cause a looping out from the
helix; and (2) strand interruption. The interferon-inducing
ac-tivity of rIn rCn is lowered by these modifications in
varying degrees; the dec.re~se is much moxe significant when
the perturbation is imposed upon the rIn strand than upon the
. ~
: ' '
; ~ ~29- .
:~ :
1 rC s-trand. This lowering of the biological activi-ty of
rIn.rCn cannot be explained by the two sim~le requirements
for thermal stability, and susceptibility to nucleases.
The Tm values of the modi-Fied rIn rCn complexes are only
slightly less than that o~ rIn rCn and are well above the
incubation temperature o~ the cells~ Ternary complex
formation with poly L lysine significantly increases the
Tm oE these polynucleotide helices~ but has little effec-t
on their biological activities. These modified complexes
are more susceptible to nucleases: however, modified
rIn rCn complexes can be prepared which have biological
activities comparable to those of the original rIn-rCn,
but are much more susceptible to nucleases. Xn addition,
ternary complex formation with polylysine (both L and D ~. -
forms) virtually protected all these polynucleotide helices
from nuclease, yet their biological activities were not
changed significantly. These observations and reasonings
indicate that the structura2. modifications introduced here :~
may be directly related to the structural requirement of
receptors in the cells responsible for the triggering .
mechanism of .interferon production.
' .`
, , ' ' .
,~
, ~ .
. ~ -29A-
..
1 The ~pparently stringent req-lirement for the
polynucleotide complex in order to be an effective interEeron
inducer is well known. However, it has been reported that
ternary complex formation with DEAE-dextran significantly
enhanced -the biological activity of rIn-rCn. Most of the
effec-t was attribu-ted by the investigators to an increase
of uptake and protection ~rom endonucleases. It has been ;~
proposed that the change of mass to charge ratio may
acilitate the uptake process, which is paxt of the
consideration related to the question of specificity.
; However, the fate of such a complex inside the cell has not
been discussed. It is not known in what manner the
DE~E-dextran can be dissociated from the rIn rCn complex or
can be degraded. Similarly, in this investigation, ternary
complex formation with polylysine ~both ~ and D), did not
~; chanye the biological activity of rIn rCn as interferon
inducer. A relevant study on the physico-chemical properties
~, . .
of DNA, RNA, rAn rUn and rIn rCn upon complex formation
has been reported. The circular dichroism spectrum Oe
rIn rCn has been drastically changed upon complex formation
with poly L lysine and the ternary complex has al50 been
investigated by high-angle X-ray diffraction, low-angle
X-ray dif~raction and by electron microscopy. While definite
information about the helical structure cannot yet be
obtained, it is most likely that the ternary complex is a
multiple~stranded fiber. It has also been reported that
the inEectious RNA of two equine encephalitis viruses lost
their infectivity upon compIex formation with poly L lysine,
though the activity of such a complex can be recovered after
. .
pronase treatment or dissociation by strong salt. This
polylysine-RNA complex was resistant to inactivation of
the nuclease.
'
~ -30-
1 At presen-t, we have no knowledge about the fate of
the polylysine-rIn rC complex outside the cell duri~g the
incubation period of one hour, or of the complex remaining
~ithin -the cell at the end of exposure aft.er washing.
It does not appear to be a simple, easy process for the
removal o~ polylysine from the ternary complex, either by
dissociation or by degradatlon (especially the D-analog).
It remains a possibility that the "receptors" in the cell
can be triggered by the entire ternary complex containing
both polylysine and rIn-rCn. However, as described above,
the conformation of rIn-rCn has been greatly changed by
the association with polylysine.
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1 L~1849
An analysis o~ thc composition revealed that the actual
synthesis produced polymers which have about one half as mucl
as the 2'-Q-MethylinOsinic originally put into the reaction
mixture. For example, in the composition given in Tables VIII
and IX, these compositions were given, as indicated in the
footnotes of those tables, as an input concentration into
the enzyme mixture during the preparation procedure but the
analysis of the result indicates the amount of mI is only
half of that put in. Therefore, the correct chemical composition,
in the cases determined, always contains half of the mI as put into
the original en~yme mixture. For example, rI10,mI which is
now cited in Table VIII represents the inpu-t ratio. The
chemical ratio of the product actually is rI20,mI, which is
footnoted in Table VIII.
A biolo~-cal activity of poly 2'-0-Methylinosinic as well
as the hybrid strands containing both iOsinic residue and
2'0-Methlinosinic residue were tested on human cells. These
-~ compounds first will form complex with rC polyribocytidylic in a
manner descri~ed in the experiments. The results indicate clearly
that the poly 2'-0-Me~ylinosinic polyrib~cytidylic 1 to 1 complex
has little or no activities as human cells interferon inducer
as compared to the original rI rC. However, in the case of the
hybrid molecules, the resultsare totally different. As recorded
in Tables VIXI and IX, the human cells and cultures respond to the
complex rC as well as the hybrid molecules of polyinosine and
2'-0-Me inosine produce significantly much better results in
tests against viral attacks as compared to the original rI-rC.
It i5 to be noted from the footnotes of Tables VIII and IX
that the composition of the products are certainly not identical
to the input of the oxiginal substrates in the enzyme preparation.
; It can be summarized fr3m Tables VIII and IX that 1 to 1
complex of rC and polyinosinic containing 5 to 16% 2'-0-Me
inosine can be 100 fold more as effective as rI^rC in inducing
-31-
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~nterferons from h~unan cells. This is a very important finding,
~perhaps allowing the administration of -the drug a hundred fold
le~s in a dose level producing the therapeutic value.
. :
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; -34- `
~ , . . .. .. .. . . ....... .. . . . . . . ..
