CA1296663C - Arabinonucleic acid probes for dna/rna assays - Google Patents
Arabinonucleic acid probes for dna/rna assaysInfo
- Publication number
- CA1296663C CA1296663C CA000525772A CA525772A CA1296663C CA 1296663 C CA1296663 C CA 1296663C CA 000525772 A CA000525772 A CA 000525772A CA 525772 A CA525772 A CA 525772A CA 1296663 C CA1296663 C CA 1296663C
- Authority
- CA
- Canada
- Prior art keywords
- probe
- dna
- acid
- arabinonucleic
- label
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
Abstract
ARABINOUNUCLEIC ACID PROBES FOR DNA/RNA ASSAYS
ABSTRACT
A novel nucleic acid, arabinonucleic acid, is provided as a probe in nucleic acid assays. The anti-arabinose antibody-label conjugates.
ABSTRACT
A novel nucleic acid, arabinonucleic acid, is provided as a probe in nucleic acid assays. The anti-arabinose antibody-label conjugates.
Description
2~6~6~
TITLE IP-05~9 TECHNICAL FI~LD
Thi6 inYention i~ rela~ed ~o arabinonueleic acid and the u6e of thi~ novel ~olynucleot~de pzobe l~
~NA or RNA hybrid~zaeion a6~ay6 ~nd e6pecially ~o probe~ con~aining a binding 6ite in every ~on6t~tuent nucleotide. Thi~ per~it6 the at~achment of ~ ~etection ~oiety to each nucleot$de of ~he probe and ~hu6 leads ~O ~mproved deeectlon sensitlvi~y.
BACKGROUND ART
Hybridization probe6 u6e~ in DNA an~ ~N~
as~ay6 are labeled i~ ~ome fa6~io~ ~o ~cili~at~
detection o~ the duple~ aft~r the probe ha~ hybri~ized with a complementary ~trand o~ ~ar~e~ DNA or ~N~ ln the ~ample under ~nalyBi6~ Mo~t ~ommonly, the probe can be label~d by enzy~atically in~orporating r~dio-labeled ~ucleotide~ on ~he 3' or 5' ter~inu6 o~ the probe. tA.~. Maxam et al., Me~hO Enzy~ol. Volu~e 65, 499-560 (19~0)~. AlternatiYely~ higher level~ o~
radiolabeled nucleotides can ~e in~o~porat~ by ~
tran61ation tP.~.J. ~gby ~t al., J. ~ol. ~lol. Yolu~e 113, 237-251 (1977)1. Thi~ er ~hod ~o~e~e~
the inh~r~nt advantage o~ b~in~ abl~ to ~ncocp~x~te one radioactiv0 ~ho6phoru~ per ~on~ti~uen~ ~ucleotide ln the probe and ~hu6 as~ay~ u6in~ the6e probe~ ~re ~harac~0rizsd by the ~06t ~n~i~iv~ de~tion lioit~
in hybridization a~say~. The obviou6 di62dv~a~e~ of the~e prob~g are the hazard~ and inconv~nien~
associat~ wi~h the u6a o~ radioiso~op~6.
A ~e~ond type of label in~olv~ 0nzy~atl~11y 6ynthe6izing ~ probe with a ~$x~ure o nucl~otide~
containing a biotinylated pyri~idine ba6e (ur~cil or ~d~no~ine) incorpo~ated in~o one o~ ehe nu~Ieo~idQ6 ~ .
te.g., P. ~. Langer ~t al., Pcoc. ~atl. AGad. &c~ 5A
Volu~e 7~, 6633-~37 (1~81)~. Alternati~rely, biotin-ylated hi6tone Hl protein6 can be chemically cro~-lirlked with ba6es in DNA to yiela ~ 13bele~ probe S lM. P~e~z, E~SB0 J. Volums 2(i), 017-822 (1983~1. Ater hybridization of the probe ~o a complementary ~trana o~ target DNA. the hyb~id ~i~ trea~ed with a ~epo~ter molecule attached to aYidirl which bind~ tenaciou~ly ~o the ~otin moiet~e6 ~a ~he pro~e. ~he repor~er can be 1~ eitbe- all opaque poly~eric ~D~cro6phere bound ~o av~din ~0. C. Richard6 et ~1., Proe. Natl. Acad. ~c~. USA
Volu~e 76(2), 676-680 (1979~] or avidl~-~erriti~
t~- Sod~a ~t ~1., Nucl. P.ela6 P~e~. Volu~e 5(2),, 3~5-~01 ~197~) ] . Each o~ ~he~e can ~e vi6ualized ~
15 ~lectron ~sro6copy. Alte~nat~v~ly, the ~eport~r ~ay be an avidin-enzyrae complex which whe~ treatea w~th the er~zy~e ' 6 ~ub6tr~te, will yield a ~rieually t~t8C~-able colored product tJ. J. Leary et ~1., Proc. Natl.
Acad. Sci. USA. Volume 80 4045-4049 (1983)]. Afi6ay6 20 based on these probe~ obviate l;he Ul;9 of radioactiYe ateri~16: howe-ver, only About 2~ o~ nuclooti~e6 in ~ given probe ~Role~ule can be deri~rat4zed ~ith a biotin ~oi~ty withou~ reducing the specif icity o~ the probe for it~ comple~Qnta~y s~ran~ of sa~pl~ DNA. Thi$
25 reduction in ~peciîici~y ari~6 fro~ ~odific~ion o~
~che 2bili~y o$ comple~e~lt~ry bags~ to ~ir prop~rly because og the pr~sence of the blot~n aoiQty~ The reduced 4ncorporation of l~bel in the~e ~robe~
relativ0 to ra~ioactive probes ro~ul~ in ~ ~oorer 30 detection 6en6il~ivity,.
A third ty~e o~ label ~5 ~n ~nzy~e directly ~ttached ~o the ~robe ~uch tha~ ~te~ b.ybri~i2ation the enzylDe in the hybrid ~ ~ ~reated with ~ub~trate to yield a colored product. The ~nzy~ne can ~e bonaed 3s directly to ba6e& in the probe tP~. ~enæ ~t al., Nucl.
~.;29~
Ac~d6 ae6., Volume 12(8), 3435-3444 (1g8~J or ~o ba~es in another ~trand of DNA ~omplementa~y ~o 2 leng~h of oligonucleotide c~em~cally attached tb tbe ~nd of the probe tJ. G. Woodhea~ et ~l~o Biochem. Soc., Trang., 5 ~olu~e 12(2), 279-~o ~1984)]. I~ the ~tt0L ~a8e, th~ ~robe 16 hybridized to complementary tar~et DNA
and thi~ hybri~ ~ treated wi~h the ol~gonucleot~de containing the enzyme label6. Thi6 ~trand o labeled oligonucleotide binæs with ~he single-~randed tall o~
10 ~omplementary DNA which had bee~ chemically attached eo the ena of ~he probe. Visualizatio~ o~ the ~nzy~e reporter6 t6 accomplished by ~onver~io~ of the enzyme'6 ~ubstrate to colored p~oduct. The pri~ary ~iffi~ultie6 a6~0ciated with ~hi6 proceau~e ari6~ from the low level of enzyme attachm~nt and the lo~ o enzy~e activa~y when ~ubjected ~o t~e ~t~ingent condition~
(eOg. elevated temperatur~, nonaqueous ~ol~ent) ~ypi-cally u6ed i~ the hybridizætion pro~ocol. A ~econdary dificulty a660ciated with the la~er o~ the~e labeling ~ethod6 i6 the po66ibility ~hat the 6ynt~etic 6tr~nd of oligonucle~tide containi~g the ~nzyme l~bel ~ight be comple~ent~ry to a 6e~uence o naturally-occurring nucleo~ide6 in the ~ample. Thus, i~ additlon to pairing wi~h the ~eque~c~ o~ b~e~ ~ttached ~o th2 end of the hybri~izatlon probe, the enzyme-lab~led oligonucleoti~e ~y bind non~pe~ically to natur~l ~equence~ of comple~ntary ba~es a~ld thus l~d to backgroun~ problem6 i~ ~ample~ u6ed a~ ~eg~tive controlfi or bl~nk~.
A ~ourth typ~ o lab~l in~olve~ coupling a ~luorophore either to b~ e6 ~n the con~tituen~ nu~leo-tid~ of ~he pro~e IC. H. Yang ~t a~ . B~oche~.
Volume 13. 3615-3~20 (1974)3 or to ~be 3'-~@r~inu6 ~R. ~. ai~hard60n et al.~ Nucl. A~ Re6. Volu~e 11(8), 6167-6184 (1983)] or to the 5I ter~inus 6~6~
~C. H. Yang et al., Arch. Bioche~. Biophye., Volu~e 155, 70~ 973~1 of ehe probe. As above, the ~en~i-~vi~y o~ the6e method6 ~6 limit~d by the ~all number of label~ whic~ can be incorporat~d into ~ach copy o~
5 the probe.
A fifth type of label involYe~ ~he use of a~
ant~body directea against 60me ~ntigenlc deter~ina~
in the probe or in t~e targe~-probe duplex. I~ the ~ormer ea8e. an antigenic determ~n~nt (e.~., bioti~, lQ bro~ine, ~-aceto~y-N 2-acetylamino~luorene) ~
covalen~ly coupled to ba6e6 i~ t~e ~u~leoeide ~, ~he probe te.g., L. Manuelidi~ o~ al., J. Cell ~iol.
Volume 95, 617-625 ~1982)]. ~he6e antigenig ~et~r-~inants ~ay iDterfere ~it~ speci~i~ hybr~dization a~d ~5 limit ~he util~ty of t~e probe. In the lat~er ca~, ehe double-6tranded DNA-RNA hybrl~ itsel~ lfi i~uno-logi~ally dis~ingui~able ro~ DNA-DNA and RN~-~NA
duplexe6 ~G. ~. Rudki~ et al.~ Nature, Volume 265.
472-~73 (1977)~. The ant~genic de~erminant ia the probe-target duplex was detected with an a~tlbody-fluorophore con~ugate ~nd fluor~6cenc~ ~icro~aopy.
The lab~led probe can ~l~o ~ dete~ted with antibodie6 to which ~n en~yme ~L. Manu~lidi~ o~ ~1., op~ ) 0 ~olloidal ~ol~ tN. J. ~utchi~on et al., J. Csll ~$ol./
Volume 95, 609-618 tl982)] has b~e~ ce~ugat~d. A~ai~, the ultimate senEiti~lty i~ ited by the nu~r of abelB av~ilabla to detect a hybridizatio~ o~e~t.
~ he infi~ant invention ove~co~e6 ~he limit~tis~
of th~ prior ~r~ ~y incorpor~ti~g ~he ~axi~al ~u~bQr o2 nonradioactlve la~el~. o~e per nucleoti~e, ~nt9 ea~h probe wit~ negllg~ble e~f~ct on hybriaiz~io~
~fficien~y or 6pe~ifi~ty.
6~i3 DI SCLOSUR~: OF TH~ I~VENTION
Thi6 invent~on involve6 a new nucleic acid, arabinonucleic a~id.
The me~t~oa of thi6 inv~ntion for iderltifying rlucleic acid 6equences s:omprise6 the B~ep~ o~:
(a) rendering the ta~get ~u~leic aci~
6ingle-~tranded:
(~ immobll~z~ng ~he 6i~agle-st~an~ed nucl~ic acid6 onto a 6upport:
10 ~c) allowing 6aia single-6trandea r~ucleic acid6 to hybrldize with i 6ingle-~tranded arabinonucle~ ~cid probe:
) wa6hing 6aid 6UppO1~ ~0 r~mo~ ~rabil~o-~ucleic acid not incorporated ~n~o 'clhe ~yb~ ormed oal the 8uppo~t: ~a ~e) determin~ng l:he pre~enGe of arabinonu-elei~ acid i~ the hybrid orDIea o~ tbe 6upport by conta~ting i~ ant~-arabino~e antibody-label conjugate ~nd detecting ~aid la~el.
DE S CR 1 PT 1 ON OF THE I NV~NT I O~N
Thi6 inv~antio~ volve6 th~ ~yn~hesi~ ~d u6e o~ ~ novel hybridization prob~ ~or DNA ~nd RNA ~ y~.
25 Thi6 new probe. arabinonucleic aci~ (ANA), IIAB ~n arabino~e sUgar rQ~l~cing ~he sor~v~ion~l ~ibo6e or deoxyribo~e 6ugar~ found i~ RNA arld ~dA, re~p~c~iv~ly.
~hi~ uncommon cugar provi~e~ b~ ling ~ito~ ~or ~ anti-arablno~e ~ntibody-label which ca~ b~ us~a to aetect 30 seleetively ~he ar~bino6~ sugar ana thu6 any ~robe containing the s~ugar. Th~ ~u~ar arabinos~ 1~ susl~
only ~n th~6 syntheti~ p~o~e and ~o~ ~n ~ y ~atur~lly-oocllrring DNA or RNA~ ~nd. tberefore, the dete~ion ~ntibody will bind only eo the ~rabino6e ~11Oiet~:26 ~t~
35 the probe.
, , . , , ..
, : ' , ~ ., ~ he probe can be 6ynthesized either ~hemically or enzymatically. Chemic~l 6ynthesis involves the ~ntroduction of protective group6 on the 2', 3' and 5 carbon a~o~6 of the arabino6~ ~ugar ~n ~ ~uGleosi~e 5 ~ontainiDg arabino6e in6~ea~ o~ ribo6e or deoxyrl-bo6e. Such nucleo~ide~ are commer~ially available.
The chemical nature and ~e~hod of a~tachi~ the pro~ectiYe group~ ha~ been develope~ ~or ribonucleo-6ide6 and de~xyrlbonucleo6ide~ tG. ~. Hakimeliah ~t 10 al.~ Can. J. Chem., Volume 60, 1106-1113 (1982)J and can be adap~ed for ~rotec~lon o~ the corre6pond~ng po6itlon6 in arab~nonu~leo~lae~ 1~. K. Og~lvi~ ~ al., Can. J. Chem., Volu~e 61, ~204-1212 ~19~3)~. O~ce the pro~ec~on of the ara~nonucl~o6~qe~ has ~een achievea, the chemical fiynthesi~ o~ t~e arabinonucleic acia probe c~n then p~o~eea in the ~annar u~ed fo~ DNA ana RNA probe6. in that the ~orrec~ nu~l~o~ide6 wlll be 6eguentially lin~ed to form ~ chai~ of nu~leotide~
which 1~ co~ple~entary ~o a ~equence of nucleotide~ in zo tar~et DNA or RNA in the ~ampl~ under analy~i~ (M. H.
Caruther~ et al., ~n Gen~tic ~nsi~2eri~g, ~. Setlo~, : ed.~ Vol. 4~ 1-17, 1982).
Nucle~ a~i~B to b~ analyzsd ha~e ~a~y ~ource~. These incluae ~lin~c~l ~peci~en6, ~ariou~
~icroorgani~ such as b~cter~a~ viruse~, ~hl~mydia, rickett6ia, ~ycopla~ma ~nd protozoa. plants, ~ong other~. Extraction 1~ one common ~ethod Pol collectin~
the nucleic ~cid~ fro~ the~r ~ourc~ ~or hybr~iza~lon ~66ays. The ~oto~ol for use of ~h2 ~NA probe~ o~ thi~
inventio~ i6 ~uch like that ~n ~onventtonal hybriaiza-tion proce~u~e~. The taLget DNA or RNA i~ ~ir~ ren-dered 6ingle-~rand0d and th~n i~mobiliz~ o~to ~
~uppor:t. The i~obilizea single-6tranded nucle~c ac~d6 ~re ~hen treated~wi~ the arabinonucleic a~id ~: 35 probe complementary to ~e~uence of ba~e6 in the ~, ` ~
~Z96G6;3 target. Hybridization of ~he arabinonucleic ~c~
probe with the complementary 8equence of bases ~
allowed to occur. Ater washing to semove exce36 unhybridized ANA probe. the probe-target hybr~
treated with an anti-arabino6e antibody-label ~on~u-qate.
The antibody it~elf i~ raified against ~he C2 chiral ~ite in arabino6e or t~e C2 ~ite in con~unct~o~
wi~h other antigenic determinant~ in the a~a~ino8e or 1~ ~NA 6tructures tOkabaya~hi e~ ~1., Cancer Re~earc~
Volu~e 37, 61g-624 [1977)1. The antibody-co~ugate can be prepared by con~entional ~ean6. The label ~an be enzymat~c~ fluorescent, ch~milu~ine6cent or ~acro-molecular, the preferEea one being ~nzymatie.
Thi~ antibody-la~el con~ugate 6pecif~c~11y bind6 to t~e arabino~e ~ugar but not ts ribo6e or deoxyribo6e pre6ent in native %NA or DNA. After treat~en~ 6uch as wa~hing to remove exces6. unbound antibody-enzyme conjugate ~rom the ~y6te~ it ~
2~ treated with ~he enzyme ' 6 6ub6trate ~o produce a detectable ~ignal such a6 a colored product, indi--~ating the pre6ence of tha targe~ nucleic aci~ i~
the ~ample. Labels other ~han enzyme6 ca~, of cour~e, be detected app~op~ Qly.
Altsrnat~v~ly, one can envi6io~ othor ~indi~
r~agent6, euch a6 a l~ctin, which could be oon5u~a~d eo an enzyme or oth~r lab~ls. The a~say protocol for ~uch ~ con~ugate would be similar eo that ~ove, ~rabinonucleic acid probe6 hav~ ~e~eral unique aavantag26. Since one arabino~e bin~ing ~t~
~an be incorporated inSo ~ach nucleotide of tb~
hybridization probe, each nucleoti~e ~6 capa~le of hav~ng an anti-arabino6e antibody-label bouna to lt to achieve labeling at the 12vel6 usually ob~ainable with radioi~otope~. At the ~a~e ti~e, the ~azara~ o~ ra~io-;
, i6~
a ac~ive material6 are avoi~ed. I~ addition, the6e fullyla~eled probe6 will not un~ergo the ~egradation that radiolabeled probeL experien~e due to 1YBi~ by the h~gh-energy decay product~ of the radiolabels. Further-~ore, since detectability o~ the AN~ relies upo~ ~heinverted chirality aboue thQ C2 carbon atom in the sugar backbone of the probe, ~nd ~ince th~ che~cal bond6 about the C2 earbon a~om ~re ~ot involved ~
determining ei~her ~he intra- or ~nter-~trand Etructure of the probe or the probe-tar~et hybrld, specifici~y of ~he probe and the stabili~y o~ the probe-target hybr~d are not expec~ed to be ~ffe~tea ~ignific~tly. This higher degree of labeli~g o ~he ~NA probe. achieved without altering t~e 6pecificity o~ the probe ~or i~6 complementary ~eguence of ba~e6, ~ak~ the probe6 of thi6 invention ~uperior to ~he ~he~i~ally aerivatized probe~ sf the prior art. Finally, the arabino6e in the probe can be speci~ically det~c~ea u6ing ~n antibody-enzyme ~on3ugate. The advantage o~ the use of an enzyme label derive~ from th~ high ~gree o 6ignal magnification due eo ~he large t~r~over o~
~ub~trate to produ~t. ~hUfi, U8~ 0~ antibody-enzy~e con~u~at2s to ~e~ect ~rabino~e po66e6ee6 ~ot only the advant~ge o~ the high ~egr~e o~ pro~e labeli~g characteristic of rAdiolabel~d probe~ but al30 ~he ~ign~l maqnif ication o~ an enzyme-ba3ed nonradio~r~
a6~a~.
B
. .
~Z~6~3 Exampl~_~
. Chemical SYnthe61~_of Protected Arabinonucleoti~es for Probe Synthesl6 Probe 6ynthe~i6 through chem~cal polymeriza-5 tion o nucleotide6 cequi~e the avail~bility of ~tlea6t four protected and activated arabinonucl~oti containing the ba6e6 guanin~, cy~o6ine, adenine, ana ura~il or thy~ine. Each o~ the6e protectea nucleo-tide6 can be prepared from the ~orre~ponding nucleo-~de~, whiCh are commercially availabl~.
~ he addition of protective ~nd activatinggroup6 ~v a nucleo~ide will be ~11UBtrated ~or the preparation of the protec~ed and activated nucleo~ide arab~nouracil tarav). The other ara~inonucleo~iaes were protected and activ~tea ~n 9~ i~entical ~a~ner:
in add~t~o~. nu~leo6ide~ ~ont~iniag the ba~e~ eyto6i~e and adenine ~equired proteetion o~ the base ~t6el~ ~n t~e form of ~ benzoyl group while guani~e ~equired D~
i~obutyryl pro~ecting group.
Fir6t. a di~ethoxytrityl (D~T) group wa6 introauced at tbe hydroxyl on the 5'-~arbo~ o~
arabinose. ~hi~ wa~ done by addi~g 7.75 ~mo~ of dimethoxytrltyl chloride (DMTCl) i~ ~igh~ equal por~
tion6 at l-hour ~nterval6 to 6.5 m~ol~ o~ ~-D ~rabino-uracil di~aolved in 30 m~ pyri~ine ~t -5~C. On~ hour a~t~r the ~inal ~ddition. the reactio~ ~ixtur~ wa~
pour~d into ice wat~r to de~troy exc~ di~etboxy-trityl chloride and re~ucea to ~ ~um ~ ~pproximat~ly 250C on a roeary ~vaporator. The product, 5'-D~T
~raU, wag i601at~a fro~ t~e r~action ~ixture o~ a eolu~n o~ Mer~k Xie~elg~l ~0 ~ a g~l elutQa with g5/5 ~ixture of ~hlorofor~ethan~ ld~ o~ 90-~5 ~ere obtained.
A;ter~-butyldi~ethy~ilyl 9lOUp (TBD~) wa~
~hen introduced 2t th~ hydroxyl ~roup OA th~ carbon : 9 '~....
6~
of ~rabino~e ~n ~he ardb~nonucleo6i~. 5'-DM~ ~ra~, 5.5 Dmole6, prepareA ~6 ~boYe, W3B ~i6601ve~ ~n ~e-thoxyethane (1~0 ~L) ~nd ~4 ~mole6 of tr~ethyl~ne ~a6 ~ded. Next. 16.5 ~mole6 o~ ~lver n~trat~ ~a6 added an~ the mixture ~tirr~ for 1 h~ur ~t roo~
temperature. Then, 16.5 mmole6 of t~rt-bu~yl-di~ethyl611yl ~hlor~e wa6 ~d~ed and ~h~ r~actio~
~ixture sti~red or 5 hour~ a~ Eoom temperature.
~e reactio~ ~xture wa6 f~ltered into ~ 10% ~olut~o~
of ~odiu~ bicarbonate ~nd tbe ~queou6 ~ixture wa~
extr&~e~ twice ~it~ ~ethylene ehlori~@. The org~nic ~xtrac~ wa6 evapor~ed to dryne~6 ~n~ 5'-DMT~3'-TBDMS
~raU wa6 1601~tea i~ 75~ yiel~ fro~ a ~ex~k Kie~elge~
60 6ilica gel cQlu~n elutea w~t~ yl ~e~ate.
A ~enzoyl (Bz) ~rotecti~ group w~ introdu~ed at the hydroYyl on t~e 2'-earbo~ ~P ~rabino6e i~ the nucle~6ide by ~16~olving 5.~ ~m~1~6 0~ the previou~ly preparea ~ MT,3~-TBDMS araU in ~9 ~L of pyridine and cooling ~o -~5C. An ex~e6s (6.6 mmol~ç) o~ benzoyl chloride in methylene chloriae wa6 ~daea dropwi~e to the ~t~rrea ~action ~ixture ~hi~h ~as beld at -~S-C
for ~0 ~inute6 after addi~ion W~6 co~plot~. The ~c-eio~ ~ixture W~6 wa~ed ~n~ h01~ ~t -20-C ~or 2 ~our~.
~fter addit~on o~ ~t2r ~o ~ydrolyz~ unre~cta~ be~zoyl chlori~e, the ~xture was r~duc~A ~o a ~u~ o~ ~ rot~ry evapor~tor and 5~-DM~. 3 ' -TBDMS, 2 ' -Bz a~U w~ olat~d on a ~lic~ ~ol ~olu~n ~lut~ w~t~ ~ 50/50 ~x~ure o~
toluene ~nd ~thyl ~cet~te.
The te~porary block~n~ ~roup (TsDM~) on ~he hydroxyl on t~o 3~arbo~ va~ r~mov~ by gro~ting 0. 7a ~molo o~ 5~-DMr~3~-TBDMs~2~-~z ~r~V w~e~ 2.5 D~ol~ of lM ~etrabutylam~on~u~ 1uor~e (~ TH~ ~t 25-C ~o~
0.5 bour. ~e 5'-DM~,2'-~2 ~raV ~a~ ~ola~ rOD
~i~ica gel column eluted with ethyl ~c~t~.
* denotes trade mark ~0 ~z~66~
1~
~ h0 ~inal step in ~he prepara~ion of the nucleotide involved the acti~at1on of the hydroxyl group on t~e 3'-~arbon wi~h a pho6phine. 5'-DMT,~ z ar~ 77 ~mole6) wa~ di~601ved i~ ~Z3 ~ o ~ethylene 5 chloride containing 48 ~L ~ ~iisopr~pylethylami~e.
Nex~, 31 ~L o~ N,N-dii60propylmethylphosphonami~c chloride wa6 added Yia 6yr~nge ~o the s~irr~d ~eactio~
~ixture at 20~. After 15 ~inu~e~, the rea~tion ~
~ure wa~ diluted ~ith ethyl acetate and ex~racted with 1~ agueou6 60dium b~carbonate t~atura~ed). The o~gani~
~ayer wa~ 6eparated ~ro~ the ~qu~ou6 bi~arbonat~. dried with Na2504 a~d redu~ea on ~ ~otazy ~vapo~ator ~o y~eld the de6~ed actiYated and protected nucleo~i~e.
15 B. Pro~e Svnthe~i~
It i6 envi6ioned ~a~ ~ynthe6is of ~ oli~o-~eric ANA probe will follow the procedures ~eveloped for produc~ion o~ DNA probe u~i~g ~i~ila~ly pro~ected and acti~ated deoxynucleotides t~. H. Caruther~, 0~
z~ al~, in Genetic En~ineerinq, ~. Se~low, ~. Volu~e ~.
pp. 1-17). According to ~he6e ~roc~dure~, the t~r~t 6~p in the 6ynthe~i6 woul~ ~e th~ aadit~o~ o~ ~
~tarter ~erivatized arabino~ucl~o~ to a ~ nol-der~vattze~ Bilica support~ Thi~ wlll be ~oa~ by Z5 roactlng the appropriate 5'D~T,2'-~z ~rabinonuclQ
~ide ~ith ~uc~inic anhydrid~. The ~uccinyl~a arabinonucleo~i~e would b~ ~o~verte~ to the p-ni~ro-phenyl ester by reActlon with p-nit~ophenol ana ~y-clohexyl earbodiim~d~. Th~ ~G~iYated ~ucl~06id~ will be reacted wi~h a~inopropyl-deriv~iz~d ~ c~ g~
~ ixture ~ ethylfor~amide, d~ox~e, ~nd triethyl-a~ine. Unre~cted 6ilanol ~roup~ o~ the sil~c~ will be blocked ~y reaction wi~h acetic anhydride. The6e ~p~
~ould re6ult in the sil~ca gel having ~he nucleo6ide a~tached ~o it a~ tb~ 3' 2nd and, therefore, t~i6 ~1 ,, .,, ~, . . .. .
. -66~
nucleo6ide will be at the 3l-end of the probe to be ~ynthe6izea.
The next nucleotide can ~hen be added to the nucleo6ide attached to the ~ a suppo~t. ~he 8il~C~-nucleo6ide produ~t prepared above will be treat~ withp-toluene6ulfonic acid in acetonitril~ to re~ov~ ~he acid-labile dimethoxytr~tyl group from the hydloxyl group on the 5'~arbo~ atom of ghe nucleo6ide ~tached to the ~ilica ~upport. The nucl~o6ide on the ~upport 10 will be conden6ed wi~h the ~ppropriate arabinonucleo-tide phosphoramidite to ~dd th~ nex~ ~ucl~otido to the 5'-~nd of ~he growing probe. Thi~ rea~tion will be carr~ed out in the pre6ence of tetrazole i~ ary a~ets-~i~rile to ~acilitate tbe ~onden6atio~ rea~tion. The unrea~ted 5'-~yd~oxyl group of the arabinonucleo6i~e on ~h~ ~upport will b~ block~d by ~reatment, for 1-2 minute69 w~th a~e~ic anhydLide i~ di~ethyla~i~opyri-dine. The p~o6phite triester linkage fo~med by t~e preceding conden6ation will be oxidized to a pho6phate eçter by treatmen~ with a ~ixture o~ iodine and 2,6-~utidine in aqueoue eet~ahydro~uran ~or l-Z ~inu~6~
Thi~ ~t~p conclud~6 ~he addi~ion o~ a nucleoeid~ to the ~rowing ~robe, Additional nucleotiae6 caa be ~dded by repeating ~he ~bo~e ~action ~equence 3tarting w~th ~he removal o~ the protective group ~rom the 5'-hydroxyl in arabinose on the 5~-~nd o~ ~be probe.
Wh~ ~h~ ~ynth~ of th~ probe ~equs~e ~B
complete, the oligo~eric probe will ~Q tr~ated vi~h tri~thylamine and th$ophenol ~n ~oxane ~o convert the pho~phate trie0ters in ~ach nucleo~i~e l~nkag~ to pho6phate ~ie6ter~. The probe then vill b~ slaav~d from the 6ilica ~uppo~e by ereatment ~ith ~oncentra~ed ammoniu~ ~ydroxiae at 20C for 3 hour6. Thi6 trea~ment Will al60 re~ove the benzoyl protecting ~roup6 ~ro~
the 2~-hydroxyl group6 in each 3rabino6~ ~iety in ehe 61Ei3 probe and any protecting group6 ~r~m ~he ba6es. The oligomer~c ANA probe will be i601ated by rever~ea pha6e liquid chrorDatography and the ~lnal dimethoxyt~tyl group on the 5~-end o~ the pr~e ~ill be remov~a ~y 5 treatment w~th 20~ acet~c a~id to yield the purii~
ANA probe, which i8 ready for use in ~he hybridiza~ion as6ay .
C. HYbridi2ation P~ocedure It ~ envi~ion~d that the proto~ol ox u6e of ~NA pr~be~ would follow along th~ e6 of the ~ariou~
~ybridi~ation a~6ay~ eom~only util~z~ ~nd ~o li~
tion6 on the ~ub~titu~ion of ANA probe~ for DNA or aNA
probe6 in any sy6tem i~ o~eseen. In~tially, ehe tar-get or ea~ple nuclei~ ~cid wcul~ ~e prepared by a~y ~onvenient p~ocedure. The ~ucleic acid ~oul~ be aen~-tured to a 6ingle-~tranded ~ta~e by a~y co~ventio~al ~ean~. For example. DNA ~an be ~enatured by h~a~i~g it in an Appropriate buf er a~ 95C for 5 ~inu~e~.
Alternative~y, denatu~ation can b0 *ffected by t~e~ting DNA with 0.25 ~ NaOH ~or 10 oinu~es. I~ ~hi~ ~8~, following denatura~ion, ie i6 n~C~6~ary ~Q add a~
equivalent ~ount o~ ~cid (~.g. HCl) to neutr~liz~ ~he colution ~ontaininy the slngle-~trandea DNA. A~ ~hi~
point, ~t may al~o be neces~r~ eo ~d~u6t ehe ~o~ic str~ngth of the ~a~ple to opti~ize the binding a~
the DNA to a ~upport. It i~ ~vi~able to cool the denatur~d ~NA on ic~ to low~r tbe rate o~ renatur~tio~
o the ~ngl@-fitranded ~NA.
~he target nuclei~ acia can ~e i~o~ zed onto the ~ufa~e of a ~upport. Cl~si~ally, t~e &uppor~ o~ ~hoice ha~ bee~ ~ nitro~ellulo~e ~e~brane.
~f thi6 ~aterial 16 U~edD an ~l~guot of ta~get ~ucle~c acid ~an eith~r be 6potted onto ~he membrane o~ 810wly ~iltered through the me~brane con~ained in 9 d~vi~e ~ ~ \
lZ~
auch a6 a dot-blot or ~lot-blot ~an~rold~ Followi~g applicatlon of the target nuclelc acia to ~h~ ni~ro~
eellulo6e, the membrane i~ aried and hea~ad in ~
vacuulD oven at approximat~ly 00 for 0~5 - 2.0 ~ours S to a66ure 6eure ~ttachment to the nitroc~llulo~e.
The 6upport ~Daterial i~ rlot ne~es6ar~1y limited to nitrocellulo6e. Por example, charged nylon support6 sucA a6 Gene ~creenT" ~. I. du Pont de Nemour~ and Co~, Inc. O Wilmirlgton" I~E9 or Biotran6Tn 10 (ICN F~adiochemical6, Irv~ne, CA) can al~o be used.
The protocols de~relop~d by tbe manu~acturer~ of ~he membrane6 6hould be followea whe~ immobilizin~ nucl~ic ~c~d~ . T~i6 appl~ e~ whethsr ~h~ nu~leic ~ci~ ei~g af f ixed to the membra~e in ~ ~ot-blo~ æanifold or by 15 one oî the tran~f er protocol6 ~ ou~chern tran6~r ) commonly u6ed af ter electropho~e~c separation of ~he ~ample nucleic a~
In a different protocol, t~e ~a~et nuclei~
acid can be immobilizea by hybridizi~g it to a 6trarld 20 Of ~capture~' nucleic acid whicb i5 ~mobilized to a ~upport ~ate~ial. This capture nucleic ~cia ie ~om-plementary to a ~hort seguence o ba~ n t,h$~ targ~t nuclei~ acid arld 6p~ ically capture6 it ~rom a ~olu-tion ~chat ~nay conta~n substdntial quar.titi~ o~ o~he~
25 nucleic acid ~hic~h are of no i~nmeflia~ce inter~t.
The nu~leic aoia o~ intere~t, which ha~ been ~irmly ir~nobilized to ~ ~upport Dlateri~ a fiingle-~tranded or denatured state and i~ a~ailable for hyb~idiz~tion with an ANA prob~ ~ontair~
30 co~pler~entary sequ~n~* o ba~e~. The im~obili2ed targe~ nucleic a~ d and the ~upport ~ ne~ct ~e treated wit~ a buffer ~olutio~ con~n~g g~n~ri~
DNA (e.g., 60nicatea ~almon ~perm DN~) ~o eli~inate non6pecific binding ~itQæ ~or the 35 6pecific A~'IA probe. Typi~ally, thi6 ~er~eric DNA
~LZ~663 i6 pre6ent ~n the buffer at a concentrat~on of 10 ~g/ml ana 100 ~1 o~ this prehybridiza~ion buffe~ ~
required ~o~ each cm2 o 6upport ~aterial. Thi6 pre-hybr~dizat~on buffer can al~o contain 10% soaium 5 ~extran ~ulfate, 0.1% 60dium dodecyl ~ulfate, 50 ~ormamide, and SSPE, which i6 a ~ixture of ~odiu~
~hloride, soaiu~ pho~phate, ~nd ~DTA. In a~aition, the bu~fer can al60 ~ontain Denhardt'~ reagent, which i6 a mixture of ficoll, polyvinyl pyrollidone, and bovine 6eruD albumin.
Th~ 6upport to which the nuclei~ ~c~
imm~bilized i6 prehybri~ized ~n thi~ buffeE mixtu~ at an elevated temperature `(~.~., 37~ - 650C) rOr ~ period of time ranging grom ~everal ~3Ur~ to overni~ht in an effort to bloGk 6i~e8 on the immobilized ~ucleic acid to which the ~NA probe can be attachea non~peci~ically.
~ollowing prehyb~idization, ANA probe 1~ ad~ed to the prehybridiza~tion buffer to the ~e~ired final concentra~
tion, typically 10 - 100 ng/~L. Hybridization i6 hen carried out at t~e appaopriate elevated te~perature (e.~., 37 - 65~C) fo~ an appropriate pe~od o~ ti~e.
u6ually overn~yht.
Following hybridization, the support i~
: washed with ~ ~e~ie~ of bu~ers to ~emove ANh ~rob*
which ~a~ be non6pecif~cally attached to the ~upport.
Typically, a6 thifi ~equenee o~ wa6he6 progres~6. the concentration of the 6alt in the bu~fe~ iB reducea ~n~
the te~perature o the waB~ iB ~ncrea~ea. ~ollowing thi~ ~erie6 o~ wa~he~, the eu~por~ i~ then rin~ed wit~
the approprlato ~ufer eo re~ove any rea~ent~ fro~ ~he wa6h buffer~ that ~ay hsve a dçtri~ental etect on the activity o~ ~be ~ntibody-enzyme con~u~a~e ~o be ~ded later.
, 9fi~6~
D. Pcoduction of Antibodles Either polyclonal or monoclonal antibodie~
can be u6ed to detect arabinosç in ~he ANA pro~e mole~ules. Polyclonal antibodie6 can be produce~ by any convenient method u~ed to pr~duce antibo~i~6 to modified DNA te.g., S. Cohn and ~. W. Lieberman, J. ~iol. Chem. Volume 259~ 12456-6Z (1984)]. ~ike-wi6e, ~onoclonal antibodie~ can be produced by a~y o~
a number of pcocedure6 (H. G. Gratzner, Science Volume 218~ ~7~-5 ~1982)1.
I~ iR belieYed tha~ an arabinonucleo~de conjugated t~ a 6uitable carrier pro~ei~ . BSA~
can al60 6erve a~ the immunogen ~n antibody proauc-tion. However. t~ ~NA probe alone ~or hybridiz~a ~o ~omplementary ~uclei~ acid~ w~uld b@ preferr~d. Alter-~atively, the probe can be conjugated ~o a ~arri~r protein to 6~rYe a6 the ~mmunoge~. Okabaya6~i ~t ~1.
tcancer ~esearch ~olume 37, 619-62g (1977)1 repor~ed production of antibodie6 to l-fl-D-a abinofurano6yl~
cyto6ine (ara-C) in pla6ma. These antibodie6 ~id not cro66-react with deoxycyt~dine or ~y~idine ~hi~h ~iffer rom ara-C only in the 2~-po~itio~. ~his sugge6t6 ~ery 6trongly that a~tibodie6 o ~uit~ble 6pecificity ~or arabino6e i~ ~NA, whi~h wlll ~ot cro~-reac~ with deoxyribo6e in DNA or ribo~e i~ RN~, can ~edeveloped.
E. Pre~aration~ hg_Antibod~-Enzy~e Çon~uq~te Con~u~ation o the an~ibody to ~n enzym~ ca~
be carried ou~ by known ~ethod~. For ~xample, th~ u6e o~ glutaraltehyde to l~nk a~1no ~oup~ on the ~nzy~e and the antibody ~B a common appro~ch ~H. ~alli~, e~
al., Cancer Letter6 Volu~e 22, 163-170 (19~4)J. U~ing thi6 p~otocol, the Qnzy~e (e.g. peroxida6e) i~ ~llo~ed ~o react for 12 hour~ a~ roo~ ~empera~ure with gluear-~ ' lZ~
aldehyde. After remo~al o~ exce~0 glutar~ldehyde i~a gel ~iltration column. the actiYate~ ~zyme allowed to react with ~he antibody for 24 hours ~
4C. The antibody-enzym2 con3ugate ~B then purlfied 5 by dialy~i6, ammonium 6ulfate ~rec~pitat~o~, ~nd ~el f iltration chromatography. Con~ugation proce~ur~
u6ing ~leteroblfunct~onal ~ro661inkir~g agent~ ~uch a~
tho6e ae6cribed ~y J. W. Yr~ytag ~t ~1., Clin. Chem., Volu~e 30, ~17-420 (198~), 02 C. C. L~lar et ~
10 ~ . Che~.~ Volume 30, l8n9-l8ll ~1984) ca~ ~lxo be u~ed. I~ i6 ~xpected t~at perox~da~e. ~-galacto6i-da6e, gluco~e oxida6e, ~lkaline or aci~ ~ho6phata6e or any o~her u~e~ul enzy~e coul~ be so~3ugate~ to the ~ntibody (or ~ fragment ~bereof) and ~ub~equently u6ea to detect ~rabino6e ~n the ANA p obe.
P. Detection of ~ybr~d~ `
The hybri~ized ~ample would be incubat~a i~ a blocking buf~er whic~ con~ains reagen~6 to reduce ~on-~pecific ad60rption of the antibody-enzy~e conjugate to the membrane. Typically, ~ buff0r ~ontaining lt bovine ~eru~ albu~in i~ u~ed. Followin~ ~eat~Qnt with the blocking bu~fer, the membrane would be i~ubat~d with a~ appropria~0 antibody-~n2y~e ~o~uga~ ~or a 2~ period of ti~e su~h that the ~ntlbo~y ha6 ~n oppor-~unity to recognize a~d bind ~o ~ach arabi~o6e ~oi~ty in the stranD~ of probe ~ybrld~z~d to earget ~u~121c ~cid. ~he antibody-~nzy~e co~3u~a~e ~oul~ eon~t o~
an enzy~e coval~ntly linke~ ~o ~n ~n~lboay or ra~men~
thereo~ which ~ ~p~ciflc ~or arabinose in th~ probe.
Following bindin~ of ehe anei~oay-enzy~e con~ugat~ to the prob~. ~be ~embLan~ whi~h ~ow contaln~ con~ugate boun~ to ~h~ probe ~ybridl2e~ to the target nueleic aoid, i~ wa~hed ~gain ~i~h a 6erie~
of buffer6 ~o re~ove unbound and non6peci~ically bound .
:
6~
con~uqate f~om the ~upport. The appropriate enzyme 6ub6trate an~/or ~hromogen ~6 ln~uba~ed ~ith ~he product on the 6upport ~nd color development ~
allowed to proceed ~or a pre6crlbed perio~ of l;$me.
5 The extent oE hybridization could be ~u~nti~ea by mea6uring the rat~ of color ~evelopment or the total color developed a~ter a Bet time period.
TITLE IP-05~9 TECHNICAL FI~LD
Thi6 inYention i~ rela~ed ~o arabinonueleic acid and the u6e of thi~ novel ~olynucleot~de pzobe l~
~NA or RNA hybrid~zaeion a6~ay6 ~nd e6pecially ~o probe~ con~aining a binding 6ite in every ~on6t~tuent nucleotide. Thi~ per~it6 the at~achment of ~ ~etection ~oiety to each nucleot$de of ~he probe and ~hu6 leads ~O ~mproved deeectlon sensitlvi~y.
BACKGROUND ART
Hybridization probe6 u6e~ in DNA an~ ~N~
as~ay6 are labeled i~ ~ome fa6~io~ ~o ~cili~at~
detection o~ the duple~ aft~r the probe ha~ hybri~ized with a complementary ~trand o~ ~ar~e~ DNA or ~N~ ln the ~ample under ~nalyBi6~ Mo~t ~ommonly, the probe can be label~d by enzy~atically in~orporating r~dio-labeled ~ucleotide~ on ~he 3' or 5' ter~inu6 o~ the probe. tA.~. Maxam et al., Me~hO Enzy~ol. Volu~e 65, 499-560 (19~0)~. AlternatiYely~ higher level~ o~
radiolabeled nucleotides can ~e in~o~porat~ by ~
tran61ation tP.~.J. ~gby ~t al., J. ~ol. ~lol. Yolu~e 113, 237-251 (1977)1. Thi~ er ~hod ~o~e~e~
the inh~r~nt advantage o~ b~in~ abl~ to ~ncocp~x~te one radioactiv0 ~ho6phoru~ per ~on~ti~uen~ ~ucleotide ln the probe and ~hu6 as~ay~ u6in~ the6e probe~ ~re ~harac~0rizsd by the ~06t ~n~i~iv~ de~tion lioit~
in hybridization a~say~. The obviou6 di62dv~a~e~ of the~e prob~g are the hazard~ and inconv~nien~
associat~ wi~h the u6a o~ radioiso~op~6.
A ~e~ond type of label in~olv~ 0nzy~atl~11y 6ynthe6izing ~ probe with a ~$x~ure o nucl~otide~
containing a biotinylated pyri~idine ba6e (ur~cil or ~d~no~ine) incorpo~ated in~o one o~ ehe nu~Ieo~idQ6 ~ .
te.g., P. ~. Langer ~t al., Pcoc. ~atl. AGad. &c~ 5A
Volu~e 7~, 6633-~37 (1~81)~. Alternati~rely, biotin-ylated hi6tone Hl protein6 can be chemically cro~-lirlked with ba6es in DNA to yiela ~ 13bele~ probe S lM. P~e~z, E~SB0 J. Volums 2(i), 017-822 (1983~1. Ater hybridization of the probe ~o a complementary ~trana o~ target DNA. the hyb~id ~i~ trea~ed with a ~epo~ter molecule attached to aYidirl which bind~ tenaciou~ly ~o the ~otin moiet~e6 ~a ~he pro~e. ~he repor~er can be 1~ eitbe- all opaque poly~eric ~D~cro6phere bound ~o av~din ~0. C. Richard6 et ~1., Proe. Natl. Acad. ~c~. USA
Volu~e 76(2), 676-680 (1979~] or avidl~-~erriti~
t~- Sod~a ~t ~1., Nucl. P.ela6 P~e~. Volu~e 5(2),, 3~5-~01 ~197~) ] . Each o~ ~he~e can ~e vi6ualized ~
15 ~lectron ~sro6copy. Alte~nat~v~ly, the ~eport~r ~ay be an avidin-enzyrae complex which whe~ treatea w~th the er~zy~e ' 6 ~ub6tr~te, will yield a ~rieually t~t8C~-able colored product tJ. J. Leary et ~1., Proc. Natl.
Acad. Sci. USA. Volume 80 4045-4049 (1983)]. Afi6ay6 20 based on these probe~ obviate l;he Ul;9 of radioactiYe ateri~16: howe-ver, only About 2~ o~ nuclooti~e6 in ~ given probe ~Role~ule can be deri~rat4zed ~ith a biotin ~oi~ty withou~ reducing the specif icity o~ the probe for it~ comple~Qnta~y s~ran~ of sa~pl~ DNA. Thi$
25 reduction in ~peciîici~y ari~6 fro~ ~odific~ion o~
~che 2bili~y o$ comple~e~lt~ry bags~ to ~ir prop~rly because og the pr~sence of the blot~n aoiQty~ The reduced 4ncorporation of l~bel in the~e ~robe~
relativ0 to ra~ioactive probes ro~ul~ in ~ ~oorer 30 detection 6en6il~ivity,.
A third ty~e o~ label ~5 ~n ~nzy~e directly ~ttached ~o the ~robe ~uch tha~ ~te~ b.ybri~i2ation the enzylDe in the hybrid ~ ~ ~reated with ~ub~trate to yield a colored product. The ~nzy~ne can ~e bonaed 3s directly to ba6e& in the probe tP~. ~enæ ~t al., Nucl.
~.;29~
Ac~d6 ae6., Volume 12(8), 3435-3444 (1g8~J or ~o ba~es in another ~trand of DNA ~omplementa~y ~o 2 leng~h of oligonucleotide c~em~cally attached tb tbe ~nd of the probe tJ. G. Woodhea~ et ~l~o Biochem. Soc., Trang., 5 ~olu~e 12(2), 279-~o ~1984)]. I~ the ~tt0L ~a8e, th~ ~robe 16 hybridized to complementary tar~et DNA
and thi~ hybri~ ~ treated wi~h the ol~gonucleot~de containing the enzyme label6. Thi6 ~trand o labeled oligonucleotide binæs with ~he single-~randed tall o~
10 ~omplementary DNA which had bee~ chemically attached eo the ena of ~he probe. Visualizatio~ o~ the ~nzy~e reporter6 t6 accomplished by ~onver~io~ of the enzyme'6 ~ubstrate to colored p~oduct. The pri~ary ~iffi~ultie6 a6~0ciated with ~hi6 proceau~e ari6~ from the low level of enzyme attachm~nt and the lo~ o enzy~e activa~y when ~ubjected ~o t~e ~t~ingent condition~
(eOg. elevated temperatur~, nonaqueous ~ol~ent) ~ypi-cally u6ed i~ the hybridizætion pro~ocol. A ~econdary dificulty a660ciated with the la~er o~ the~e labeling ~ethod6 i6 the po66ibility ~hat the 6ynt~etic 6tr~nd of oligonucle~tide containi~g the ~nzyme l~bel ~ight be comple~ent~ry to a 6e~uence o naturally-occurring nucleo~ide6 in the ~ample. Thus, i~ additlon to pairing wi~h the ~eque~c~ o~ b~e~ ~ttached ~o th2 end of the hybri~izatlon probe, the enzyme-lab~led oligonucleoti~e ~y bind non~pe~ically to natur~l ~equence~ of comple~ntary ba~es a~ld thus l~d to backgroun~ problem6 i~ ~ample~ u6ed a~ ~eg~tive controlfi or bl~nk~.
A ~ourth typ~ o lab~l in~olve~ coupling a ~luorophore either to b~ e6 ~n the con~tituen~ nu~leo-tid~ of ~he pro~e IC. H. Yang ~t a~ . B~oche~.
Volume 13. 3615-3~20 (1974)3 or to ~be 3'-~@r~inu6 ~R. ~. ai~hard60n et al.~ Nucl. A~ Re6. Volu~e 11(8), 6167-6184 (1983)] or to the 5I ter~inus 6~6~
~C. H. Yang et al., Arch. Bioche~. Biophye., Volu~e 155, 70~ 973~1 of ehe probe. As above, the ~en~i-~vi~y o~ the6e method6 ~6 limit~d by the ~all number of label~ whic~ can be incorporat~d into ~ach copy o~
5 the probe.
A fifth type of label involYe~ ~he use of a~
ant~body directea against 60me ~ntigenlc deter~ina~
in the probe or in t~e targe~-probe duplex. I~ the ~ormer ea8e. an antigenic determ~n~nt (e.~., bioti~, lQ bro~ine, ~-aceto~y-N 2-acetylamino~luorene) ~
covalen~ly coupled to ba6e6 i~ t~e ~u~leoeide ~, ~he probe te.g., L. Manuelidi~ o~ al., J. Cell ~iol.
Volume 95, 617-625 ~1982)]. ~he6e antigenig ~et~r-~inants ~ay iDterfere ~it~ speci~i~ hybr~dization a~d ~5 limit ~he util~ty of t~e probe. In the lat~er ca~, ehe double-6tranded DNA-RNA hybrl~ itsel~ lfi i~uno-logi~ally dis~ingui~able ro~ DNA-DNA and RN~-~NA
duplexe6 ~G. ~. Rudki~ et al.~ Nature, Volume 265.
472-~73 (1977)~. The ant~genic de~erminant ia the probe-target duplex was detected with an a~tlbody-fluorophore con~ugate ~nd fluor~6cenc~ ~icro~aopy.
The lab~led probe can ~l~o ~ dete~ted with antibodie6 to which ~n en~yme ~L. Manu~lidi~ o~ ~1., op~ ) 0 ~olloidal ~ol~ tN. J. ~utchi~on et al., J. Csll ~$ol./
Volume 95, 609-618 tl982)] has b~e~ ce~ugat~d. A~ai~, the ultimate senEiti~lty i~ ited by the nu~r of abelB av~ilabla to detect a hybridizatio~ o~e~t.
~ he infi~ant invention ove~co~e6 ~he limit~tis~
of th~ prior ~r~ ~y incorpor~ti~g ~he ~axi~al ~u~bQr o2 nonradioactlve la~el~. o~e per nucleoti~e, ~nt9 ea~h probe wit~ negllg~ble e~f~ct on hybriaiz~io~
~fficien~y or 6pe~ifi~ty.
6~i3 DI SCLOSUR~: OF TH~ I~VENTION
Thi6 invent~on involve6 a new nucleic acid, arabinonucleic a~id.
The me~t~oa of thi6 inv~ntion for iderltifying rlucleic acid 6equences s:omprise6 the B~ep~ o~:
(a) rendering the ta~get ~u~leic aci~
6ingle-~tranded:
(~ immobll~z~ng ~he 6i~agle-st~an~ed nucl~ic acid6 onto a 6upport:
10 ~c) allowing 6aia single-6trandea r~ucleic acid6 to hybrldize with i 6ingle-~tranded arabinonucle~ ~cid probe:
) wa6hing 6aid 6UppO1~ ~0 r~mo~ ~rabil~o-~ucleic acid not incorporated ~n~o 'clhe ~yb~ ormed oal the 8uppo~t: ~a ~e) determin~ng l:he pre~enGe of arabinonu-elei~ acid i~ the hybrid orDIea o~ tbe 6upport by conta~ting i~ ant~-arabino~e antibody-label conjugate ~nd detecting ~aid la~el.
DE S CR 1 PT 1 ON OF THE I NV~NT I O~N
Thi6 inv~antio~ volve6 th~ ~yn~hesi~ ~d u6e o~ ~ novel hybridization prob~ ~or DNA ~nd RNA ~ y~.
25 Thi6 new probe. arabinonucleic aci~ (ANA), IIAB ~n arabino~e sUgar rQ~l~cing ~he sor~v~ion~l ~ibo6e or deoxyribo~e 6ugar~ found i~ RNA arld ~dA, re~p~c~iv~ly.
~hi~ uncommon cugar provi~e~ b~ ling ~ito~ ~or ~ anti-arablno~e ~ntibody-label which ca~ b~ us~a to aetect 30 seleetively ~he ar~bino6~ sugar ana thu6 any ~robe containing the s~ugar. Th~ ~u~ar arabinos~ 1~ susl~
only ~n th~6 syntheti~ p~o~e and ~o~ ~n ~ y ~atur~lly-oocllrring DNA or RNA~ ~nd. tberefore, the dete~ion ~ntibody will bind only eo the ~rabino6e ~11Oiet~:26 ~t~
35 the probe.
, , . , , ..
, : ' , ~ ., ~ he probe can be 6ynthesized either ~hemically or enzymatically. Chemic~l 6ynthesis involves the ~ntroduction of protective group6 on the 2', 3' and 5 carbon a~o~6 of the arabino6~ ~ugar ~n ~ ~uGleosi~e 5 ~ontainiDg arabino6e in6~ea~ o~ ribo6e or deoxyrl-bo6e. Such nucleo~ide~ are commer~ially available.
The chemical nature and ~e~hod of a~tachi~ the pro~ectiYe group~ ha~ been develope~ ~or ribonucleo-6ide6 and de~xyrlbonucleo6ide~ tG. ~. Hakimeliah ~t 10 al.~ Can. J. Chem., Volume 60, 1106-1113 (1982)J and can be adap~ed for ~rotec~lon o~ the corre6pond~ng po6itlon6 in arab~nonu~leo~lae~ 1~. K. Og~lvi~ ~ al., Can. J. Chem., Volu~e 61, ~204-1212 ~19~3)~. O~ce the pro~ec~on of the ara~nonucl~o6~qe~ has ~een achievea, the chemical fiynthesi~ o~ t~e arabinonucleic acia probe c~n then p~o~eea in the ~annar u~ed fo~ DNA ana RNA probe6. in that the ~orrec~ nu~l~o~ide6 wlll be 6eguentially lin~ed to form ~ chai~ of nu~leotide~
which 1~ co~ple~entary ~o a ~equence of nucleotide~ in zo tar~et DNA or RNA in the ~ampl~ under analy~i~ (M. H.
Caruther~ et al., ~n Gen~tic ~nsi~2eri~g, ~. Setlo~, : ed.~ Vol. 4~ 1-17, 1982).
Nucle~ a~i~B to b~ analyzsd ha~e ~a~y ~ource~. These incluae ~lin~c~l ~peci~en6, ~ariou~
~icroorgani~ such as b~cter~a~ viruse~, ~hl~mydia, rickett6ia, ~ycopla~ma ~nd protozoa. plants, ~ong other~. Extraction 1~ one common ~ethod Pol collectin~
the nucleic ~cid~ fro~ the~r ~ourc~ ~or hybr~iza~lon ~66ays. The ~oto~ol for use of ~h2 ~NA probe~ o~ thi~
inventio~ i6 ~uch like that ~n ~onventtonal hybriaiza-tion proce~u~e~. The taLget DNA or RNA i~ ~ir~ ren-dered 6ingle-~rand0d and th~n i~mobiliz~ o~to ~
~uppor:t. The i~obilizea single-6tranded nucle~c ac~d6 ~re ~hen treated~wi~ the arabinonucleic a~id ~: 35 probe complementary to ~e~uence of ba~e6 in the ~, ` ~
~Z96G6;3 target. Hybridization of ~he arabinonucleic ~c~
probe with the complementary 8equence of bases ~
allowed to occur. Ater washing to semove exce36 unhybridized ANA probe. the probe-target hybr~
treated with an anti-arabino6e antibody-label ~on~u-qate.
The antibody it~elf i~ raified against ~he C2 chiral ~ite in arabino6e or t~e C2 ~ite in con~unct~o~
wi~h other antigenic determinant~ in the a~a~ino8e or 1~ ~NA 6tructures tOkabaya~hi e~ ~1., Cancer Re~earc~
Volu~e 37, 61g-624 [1977)1. The antibody-co~ugate can be prepared by con~entional ~ean6. The label ~an be enzymat~c~ fluorescent, ch~milu~ine6cent or ~acro-molecular, the preferEea one being ~nzymatie.
Thi~ antibody-la~el con~ugate 6pecif~c~11y bind6 to t~e arabino~e ~ugar but not ts ribo6e or deoxyribo6e pre6ent in native %NA or DNA. After treat~en~ 6uch as wa~hing to remove exces6. unbound antibody-enzyme conjugate ~rom the ~y6te~ it ~
2~ treated with ~he enzyme ' 6 6ub6trate ~o produce a detectable ~ignal such a6 a colored product, indi--~ating the pre6ence of tha targe~ nucleic aci~ i~
the ~ample. Labels other ~han enzyme6 ca~, of cour~e, be detected app~op~ Qly.
Altsrnat~v~ly, one can envi6io~ othor ~indi~
r~agent6, euch a6 a l~ctin, which could be oon5u~a~d eo an enzyme or oth~r lab~ls. The a~say protocol for ~uch ~ con~ugate would be similar eo that ~ove, ~rabinonucleic acid probe6 hav~ ~e~eral unique aavantag26. Since one arabino~e bin~ing ~t~
~an be incorporated inSo ~ach nucleotide of tb~
hybridization probe, each nucleoti~e ~6 capa~le of hav~ng an anti-arabino6e antibody-label bouna to lt to achieve labeling at the 12vel6 usually ob~ainable with radioi~otope~. At the ~a~e ti~e, the ~azara~ o~ ra~io-;
, i6~
a ac~ive material6 are avoi~ed. I~ addition, the6e fullyla~eled probe6 will not un~ergo the ~egradation that radiolabeled probeL experien~e due to 1YBi~ by the h~gh-energy decay product~ of the radiolabels. Further-~ore, since detectability o~ the AN~ relies upo~ ~heinverted chirality aboue thQ C2 carbon atom in the sugar backbone of the probe, ~nd ~ince th~ che~cal bond6 about the C2 earbon a~om ~re ~ot involved ~
determining ei~her ~he intra- or ~nter-~trand Etructure of the probe or the probe-tar~et hybrld, specifici~y of ~he probe and the stabili~y o~ the probe-target hybr~d are not expec~ed to be ~ffe~tea ~ignific~tly. This higher degree of labeli~g o ~he ~NA probe. achieved without altering t~e 6pecificity o~ the probe ~or i~6 complementary ~eguence of ba~e6, ~ak~ the probe6 of thi6 invention ~uperior to ~he ~he~i~ally aerivatized probe~ sf the prior art. Finally, the arabino6e in the probe can be speci~ically det~c~ea u6ing ~n antibody-enzyme ~on3ugate. The advantage o~ the use of an enzyme label derive~ from th~ high ~gree o 6ignal magnification due eo ~he large t~r~over o~
~ub~trate to produ~t. ~hUfi, U8~ 0~ antibody-enzy~e con~u~at2s to ~e~ect ~rabino~e po66e6ee6 ~ot only the advant~ge o~ the high ~egr~e o~ pro~e labeli~g characteristic of rAdiolabel~d probe~ but al30 ~he ~ign~l maqnif ication o~ an enzyme-ba3ed nonradio~r~
a6~a~.
B
. .
~Z~6~3 Exampl~_~
. Chemical SYnthe61~_of Protected Arabinonucleoti~es for Probe Synthesl6 Probe 6ynthe~i6 through chem~cal polymeriza-5 tion o nucleotide6 cequi~e the avail~bility of ~tlea6t four protected and activated arabinonucl~oti containing the ba6e6 guanin~, cy~o6ine, adenine, ana ura~il or thy~ine. Each o~ the6e protectea nucleo-tide6 can be prepared from the ~orre~ponding nucleo-~de~, whiCh are commercially availabl~.
~ he addition of protective ~nd activatinggroup6 ~v a nucleo~ide will be ~11UBtrated ~or the preparation of the protec~ed and activated nucleo~ide arab~nouracil tarav). The other ara~inonucleo~iaes were protected and activ~tea ~n 9~ i~entical ~a~ner:
in add~t~o~. nu~leo6ide~ ~ont~iniag the ba~e~ eyto6i~e and adenine ~equired proteetion o~ the base ~t6el~ ~n t~e form of ~ benzoyl group while guani~e ~equired D~
i~obutyryl pro~ecting group.
Fir6t. a di~ethoxytrityl (D~T) group wa6 introauced at tbe hydroxyl on the 5'-~arbo~ o~
arabinose. ~hi~ wa~ done by addi~g 7.75 ~mo~ of dimethoxytrltyl chloride (DMTCl) i~ ~igh~ equal por~
tion6 at l-hour ~nterval6 to 6.5 m~ol~ o~ ~-D ~rabino-uracil di~aolved in 30 m~ pyri~ine ~t -5~C. On~ hour a~t~r the ~inal ~ddition. the reactio~ ~ixtur~ wa~
pour~d into ice wat~r to de~troy exc~ di~etboxy-trityl chloride and re~ucea to ~ ~um ~ ~pproximat~ly 250C on a roeary ~vaporator. The product, 5'-D~T
~raU, wag i601at~a fro~ t~e r~action ~ixture o~ a eolu~n o~ Mer~k Xie~elg~l ~0 ~ a g~l elutQa with g5/5 ~ixture of ~hlorofor~ethan~ ld~ o~ 90-~5 ~ere obtained.
A;ter~-butyldi~ethy~ilyl 9lOUp (TBD~) wa~
~hen introduced 2t th~ hydroxyl ~roup OA th~ carbon : 9 '~....
6~
of ~rabino~e ~n ~he ardb~nonucleo6i~. 5'-DM~ ~ra~, 5.5 Dmole6, prepareA ~6 ~boYe, W3B ~i6601ve~ ~n ~e-thoxyethane (1~0 ~L) ~nd ~4 ~mole6 of tr~ethyl~ne ~a6 ~ded. Next. 16.5 ~mole6 o~ ~lver n~trat~ ~a6 added an~ the mixture ~tirr~ for 1 h~ur ~t roo~
temperature. Then, 16.5 mmole6 of t~rt-bu~yl-di~ethyl611yl ~hlor~e wa6 ~d~ed and ~h~ r~actio~
~ixture sti~red or 5 hour~ a~ Eoom temperature.
~e reactio~ ~xture wa6 f~ltered into ~ 10% ~olut~o~
of ~odiu~ bicarbonate ~nd tbe ~queou6 ~ixture wa~
extr&~e~ twice ~it~ ~ethylene ehlori~@. The org~nic ~xtrac~ wa6 evapor~ed to dryne~6 ~n~ 5'-DMT~3'-TBDMS
~raU wa6 1601~tea i~ 75~ yiel~ fro~ a ~ex~k Kie~elge~
60 6ilica gel cQlu~n elutea w~t~ yl ~e~ate.
A ~enzoyl (Bz) ~rotecti~ group w~ introdu~ed at the hydroYyl on t~e 2'-earbo~ ~P ~rabino6e i~ the nucle~6ide by ~16~olving 5.~ ~m~1~6 0~ the previou~ly preparea ~ MT,3~-TBDMS araU in ~9 ~L of pyridine and cooling ~o -~5C. An ex~e6s (6.6 mmol~ç) o~ benzoyl chloride in methylene chloriae wa6 ~daea dropwi~e to the ~t~rrea ~action ~ixture ~hi~h ~as beld at -~S-C
for ~0 ~inute6 after addi~ion W~6 co~plot~. The ~c-eio~ ~ixture W~6 wa~ed ~n~ h01~ ~t -20-C ~or 2 ~our~.
~fter addit~on o~ ~t2r ~o ~ydrolyz~ unre~cta~ be~zoyl chlori~e, the ~xture was r~duc~A ~o a ~u~ o~ ~ rot~ry evapor~tor and 5~-DM~. 3 ' -TBDMS, 2 ' -Bz a~U w~ olat~d on a ~lic~ ~ol ~olu~n ~lut~ w~t~ ~ 50/50 ~x~ure o~
toluene ~nd ~thyl ~cet~te.
The te~porary block~n~ ~roup (TsDM~) on ~he hydroxyl on t~o 3~arbo~ va~ r~mov~ by gro~ting 0. 7a ~molo o~ 5~-DMr~3~-TBDMs~2~-~z ~r~V w~e~ 2.5 D~ol~ of lM ~etrabutylam~on~u~ 1uor~e (~ TH~ ~t 25-C ~o~
0.5 bour. ~e 5'-DM~,2'-~2 ~raV ~a~ ~ola~ rOD
~i~ica gel column eluted with ethyl ~c~t~.
* denotes trade mark ~0 ~z~66~
1~
~ h0 ~inal step in ~he prepara~ion of the nucleotide involved the acti~at1on of the hydroxyl group on t~e 3'-~arbon wi~h a pho6phine. 5'-DMT,~ z ar~ 77 ~mole6) wa~ di~601ved i~ ~Z3 ~ o ~ethylene 5 chloride containing 48 ~L ~ ~iisopr~pylethylami~e.
Nex~, 31 ~L o~ N,N-dii60propylmethylphosphonami~c chloride wa6 added Yia 6yr~nge ~o the s~irr~d ~eactio~
~ixture at 20~. After 15 ~inu~e~, the rea~tion ~
~ure wa~ diluted ~ith ethyl acetate and ex~racted with 1~ agueou6 60dium b~carbonate t~atura~ed). The o~gani~
~ayer wa~ 6eparated ~ro~ the ~qu~ou6 bi~arbonat~. dried with Na2504 a~d redu~ea on ~ ~otazy ~vapo~ator ~o y~eld the de6~ed actiYated and protected nucleo~i~e.
15 B. Pro~e Svnthe~i~
It i6 envi6ioned ~a~ ~ynthe6is of ~ oli~o-~eric ANA probe will follow the procedures ~eveloped for produc~ion o~ DNA probe u~i~g ~i~ila~ly pro~ected and acti~ated deoxynucleotides t~. H. Caruther~, 0~
z~ al~, in Genetic En~ineerinq, ~. Se~low, ~. Volu~e ~.
pp. 1-17). According to ~he6e ~roc~dure~, the t~r~t 6~p in the 6ynthe~i6 woul~ ~e th~ aadit~o~ o~ ~
~tarter ~erivatized arabino~ucl~o~ to a ~ nol-der~vattze~ Bilica support~ Thi~ wlll be ~oa~ by Z5 roactlng the appropriate 5'D~T,2'-~z ~rabinonuclQ
~ide ~ith ~uc~inic anhydrid~. The ~uccinyl~a arabinonucleo~i~e would b~ ~o~verte~ to the p-ni~ro-phenyl ester by reActlon with p-nit~ophenol ana ~y-clohexyl earbodiim~d~. Th~ ~G~iYated ~ucl~06id~ will be reacted wi~h a~inopropyl-deriv~iz~d ~ c~ g~
~ ixture ~ ethylfor~amide, d~ox~e, ~nd triethyl-a~ine. Unre~cted 6ilanol ~roup~ o~ the sil~c~ will be blocked ~y reaction wi~h acetic anhydride. The6e ~p~
~ould re6ult in the sil~ca gel having ~he nucleo6ide a~tached ~o it a~ tb~ 3' 2nd and, therefore, t~i6 ~1 ,, .,, ~, . . .. .
. -66~
nucleo6ide will be at the 3l-end of the probe to be ~ynthe6izea.
The next nucleotide can ~hen be added to the nucleo6ide attached to the ~ a suppo~t. ~he 8il~C~-nucleo6ide produ~t prepared above will be treat~ withp-toluene6ulfonic acid in acetonitril~ to re~ov~ ~he acid-labile dimethoxytr~tyl group from the hydloxyl group on the 5'~arbo~ atom of ghe nucleo6ide ~tached to the ~ilica ~upport. The nucl~o6ide on the ~upport 10 will be conden6ed wi~h the ~ppropriate arabinonucleo-tide phosphoramidite to ~dd th~ nex~ ~ucl~otido to the 5'-~nd of ~he growing probe. Thi~ rea~tion will be carr~ed out in the pre6ence of tetrazole i~ ary a~ets-~i~rile to ~acilitate tbe ~onden6atio~ rea~tion. The unrea~ted 5'-~yd~oxyl group of the arabinonucleo6i~e on ~h~ ~upport will b~ block~d by ~reatment, for 1-2 minute69 w~th a~e~ic anhydLide i~ di~ethyla~i~opyri-dine. The p~o6phite triester linkage fo~med by t~e preceding conden6ation will be oxidized to a pho6phate eçter by treatmen~ with a ~ixture o~ iodine and 2,6-~utidine in aqueoue eet~ahydro~uran ~or l-Z ~inu~6~
Thi~ ~t~p conclud~6 ~he addi~ion o~ a nucleoeid~ to the ~rowing ~robe, Additional nucleotiae6 caa be ~dded by repeating ~he ~bo~e ~action ~equence 3tarting w~th ~he removal o~ the protective group ~rom the 5'-hydroxyl in arabinose on the 5~-~nd o~ ~be probe.
Wh~ ~h~ ~ynth~ of th~ probe ~equs~e ~B
complete, the oligo~eric probe will ~Q tr~ated vi~h tri~thylamine and th$ophenol ~n ~oxane ~o convert the pho~phate trie0ters in ~ach nucleo~i~e l~nkag~ to pho6phate ~ie6ter~. The probe then vill b~ slaav~d from the 6ilica ~uppo~e by ereatment ~ith ~oncentra~ed ammoniu~ ~ydroxiae at 20C for 3 hour6. Thi6 trea~ment Will al60 re~ove the benzoyl protecting ~roup6 ~ro~
the 2~-hydroxyl group6 in each 3rabino6~ ~iety in ehe 61Ei3 probe and any protecting group6 ~r~m ~he ba6es. The oligomer~c ANA probe will be i601ated by rever~ea pha6e liquid chrorDatography and the ~lnal dimethoxyt~tyl group on the 5~-end o~ the pr~e ~ill be remov~a ~y 5 treatment w~th 20~ acet~c a~id to yield the purii~
ANA probe, which i8 ready for use in ~he hybridiza~ion as6ay .
C. HYbridi2ation P~ocedure It ~ envi~ion~d that the proto~ol ox u6e of ~NA pr~be~ would follow along th~ e6 of the ~ariou~
~ybridi~ation a~6ay~ eom~only util~z~ ~nd ~o li~
tion6 on the ~ub~titu~ion of ANA probe~ for DNA or aNA
probe6 in any sy6tem i~ o~eseen. In~tially, ehe tar-get or ea~ple nuclei~ ~cid wcul~ ~e prepared by a~y ~onvenient p~ocedure. The ~ucleic acid ~oul~ be aen~-tured to a 6ingle-~tranded ~ta~e by a~y co~ventio~al ~ean~. For example. DNA ~an be ~enatured by h~a~i~g it in an Appropriate buf er a~ 95C for 5 ~inu~e~.
Alternative~y, denatu~ation can b0 *ffected by t~e~ting DNA with 0.25 ~ NaOH ~or 10 oinu~es. I~ ~hi~ ~8~, following denatura~ion, ie i6 n~C~6~ary ~Q add a~
equivalent ~ount o~ ~cid (~.g. HCl) to neutr~liz~ ~he colution ~ontaininy the slngle-~trandea DNA. A~ ~hi~
point, ~t may al~o be neces~r~ eo ~d~u6t ehe ~o~ic str~ngth of the ~a~ple to opti~ize the binding a~
the DNA to a ~upport. It i~ ~vi~able to cool the denatur~d ~NA on ic~ to low~r tbe rate o~ renatur~tio~
o the ~ngl@-fitranded ~NA.
~he target nuclei~ acia can ~e i~o~ zed onto the ~ufa~e of a ~upport. Cl~si~ally, t~e &uppor~ o~ ~hoice ha~ bee~ ~ nitro~ellulo~e ~e~brane.
~f thi6 ~aterial 16 U~edD an ~l~guot of ta~get ~ucle~c acid ~an eith~r be 6potted onto ~he membrane o~ 810wly ~iltered through the me~brane con~ained in 9 d~vi~e ~ ~ \
lZ~
auch a6 a dot-blot or ~lot-blot ~an~rold~ Followi~g applicatlon of the target nuclelc acia to ~h~ ni~ro~
eellulo6e, the membrane i~ aried and hea~ad in ~
vacuulD oven at approximat~ly 00 for 0~5 - 2.0 ~ours S to a66ure 6eure ~ttachment to the nitroc~llulo~e.
The 6upport ~Daterial i~ rlot ne~es6ar~1y limited to nitrocellulo6e. Por example, charged nylon support6 sucA a6 Gene ~creenT" ~. I. du Pont de Nemour~ and Co~, Inc. O Wilmirlgton" I~E9 or Biotran6Tn 10 (ICN F~adiochemical6, Irv~ne, CA) can al~o be used.
The protocols de~relop~d by tbe manu~acturer~ of ~he membrane6 6hould be followea whe~ immobilizin~ nucl~ic ~c~d~ . T~i6 appl~ e~ whethsr ~h~ nu~leic ~ci~ ei~g af f ixed to the membra~e in ~ ~ot-blo~ æanifold or by 15 one oî the tran~f er protocol6 ~ ou~chern tran6~r ) commonly u6ed af ter electropho~e~c separation of ~he ~ample nucleic a~
In a different protocol, t~e ~a~et nuclei~
acid can be immobilizea by hybridizi~g it to a 6trarld 20 Of ~capture~' nucleic acid whicb i5 ~mobilized to a ~upport ~ate~ial. This capture nucleic ~cia ie ~om-plementary to a ~hort seguence o ba~ n t,h$~ targ~t nuclei~ acid arld 6p~ ically capture6 it ~rom a ~olu-tion ~chat ~nay conta~n substdntial quar.titi~ o~ o~he~
25 nucleic acid ~hic~h are of no i~nmeflia~ce inter~t.
The nu~leic aoia o~ intere~t, which ha~ been ~irmly ir~nobilized to ~ ~upport Dlateri~ a fiingle-~tranded or denatured state and i~ a~ailable for hyb~idiz~tion with an ANA prob~ ~ontair~
30 co~pler~entary sequ~n~* o ba~e~. The im~obili2ed targe~ nucleic a~ d and the ~upport ~ ne~ct ~e treated wit~ a buffer ~olutio~ con~n~g g~n~ri~
DNA (e.g., 60nicatea ~almon ~perm DN~) ~o eli~inate non6pecific binding ~itQæ ~or the 35 6pecific A~'IA probe. Typi~ally, thi6 ~er~eric DNA
~LZ~663 i6 pre6ent ~n the buffer at a concentrat~on of 10 ~g/ml ana 100 ~1 o~ this prehybridiza~ion buffe~ ~
required ~o~ each cm2 o 6upport ~aterial. Thi6 pre-hybr~dizat~on buffer can al~o contain 10% soaium 5 ~extran ~ulfate, 0.1% 60dium dodecyl ~ulfate, 50 ~ormamide, and SSPE, which i6 a ~ixture of ~odiu~
~hloride, soaiu~ pho~phate, ~nd ~DTA. In a~aition, the bu~fer can al60 ~ontain Denhardt'~ reagent, which i6 a mixture of ficoll, polyvinyl pyrollidone, and bovine 6eruD albumin.
Th~ 6upport to which the nuclei~ ~c~
imm~bilized i6 prehybri~ized ~n thi~ buffeE mixtu~ at an elevated temperature `(~.~., 37~ - 650C) rOr ~ period of time ranging grom ~everal ~3Ur~ to overni~ht in an effort to bloGk 6i~e8 on the immobilized ~ucleic acid to which the ~NA probe can be attachea non~peci~ically.
~ollowing prehyb~idization, ANA probe 1~ ad~ed to the prehybridiza~tion buffer to the ~e~ired final concentra~
tion, typically 10 - 100 ng/~L. Hybridization i6 hen carried out at t~e appaopriate elevated te~perature (e.~., 37 - 65~C) fo~ an appropriate pe~od o~ ti~e.
u6ually overn~yht.
Following hybridization, the support i~
: washed with ~ ~e~ie~ of bu~ers to ~emove ANh ~rob*
which ~a~ be non6pecif~cally attached to the ~upport.
Typically, a6 thifi ~equenee o~ wa6he6 progres~6. the concentration of the 6alt in the bu~fe~ iB reducea ~n~
the te~perature o the waB~ iB ~ncrea~ea. ~ollowing thi~ ~erie6 o~ wa~he~, the eu~por~ i~ then rin~ed wit~
the approprlato ~ufer eo re~ove any rea~ent~ fro~ ~he wa6h buffer~ that ~ay hsve a dçtri~ental etect on the activity o~ ~be ~ntibody-enzyme con~u~a~e ~o be ~ded later.
, 9fi~6~
D. Pcoduction of Antibodles Either polyclonal or monoclonal antibodie~
can be u6ed to detect arabinosç in ~he ANA pro~e mole~ules. Polyclonal antibodie6 can be produce~ by any convenient method u~ed to pr~duce antibo~i~6 to modified DNA te.g., S. Cohn and ~. W. Lieberman, J. ~iol. Chem. Volume 259~ 12456-6Z (1984)]. ~ike-wi6e, ~onoclonal antibodie~ can be produced by a~y o~
a number of pcocedure6 (H. G. Gratzner, Science Volume 218~ ~7~-5 ~1982)1.
I~ iR belieYed tha~ an arabinonucleo~de conjugated t~ a 6uitable carrier pro~ei~ . BSA~
can al60 6erve a~ the immunogen ~n antibody proauc-tion. However. t~ ~NA probe alone ~or hybridiz~a ~o ~omplementary ~uclei~ acid~ w~uld b@ preferr~d. Alter-~atively, the probe can be conjugated ~o a ~arri~r protein to 6~rYe a6 the ~mmunoge~. Okabaya6~i ~t ~1.
tcancer ~esearch ~olume 37, 619-62g (1977)1 repor~ed production of antibodie6 to l-fl-D-a abinofurano6yl~
cyto6ine (ara-C) in pla6ma. These antibodie6 ~id not cro66-react with deoxycyt~dine or ~y~idine ~hi~h ~iffer rom ara-C only in the 2~-po~itio~. ~his sugge6t6 ~ery 6trongly that a~tibodie6 o ~uit~ble 6pecificity ~or arabino6e i~ ~NA, whi~h wlll ~ot cro~-reac~ with deoxyribo6e in DNA or ribo~e i~ RN~, can ~edeveloped.
E. Pre~aration~ hg_Antibod~-Enzy~e Çon~uq~te Con~u~ation o the an~ibody to ~n enzym~ ca~
be carried ou~ by known ~ethod~. For ~xample, th~ u6e o~ glutaraltehyde to l~nk a~1no ~oup~ on the ~nzy~e and the antibody ~B a common appro~ch ~H. ~alli~, e~
al., Cancer Letter6 Volu~e 22, 163-170 (19~4)J. U~ing thi6 p~otocol, the Qnzy~e (e.g. peroxida6e) i~ ~llo~ed ~o react for 12 hour~ a~ roo~ ~empera~ure with gluear-~ ' lZ~
aldehyde. After remo~al o~ exce~0 glutar~ldehyde i~a gel ~iltration column. the actiYate~ ~zyme allowed to react with ~he antibody for 24 hours ~
4C. The antibody-enzym2 con3ugate ~B then purlfied 5 by dialy~i6, ammonium 6ulfate ~rec~pitat~o~, ~nd ~el f iltration chromatography. Con~ugation proce~ur~
u6ing ~leteroblfunct~onal ~ro661inkir~g agent~ ~uch a~
tho6e ae6cribed ~y J. W. Yr~ytag ~t ~1., Clin. Chem., Volu~e 30, ~17-420 (198~), 02 C. C. L~lar et ~
10 ~ . Che~.~ Volume 30, l8n9-l8ll ~1984) ca~ ~lxo be u~ed. I~ i6 ~xpected t~at perox~da~e. ~-galacto6i-da6e, gluco~e oxida6e, ~lkaline or aci~ ~ho6phata6e or any o~her u~e~ul enzy~e coul~ be so~3ugate~ to the ~ntibody (or ~ fragment ~bereof) and ~ub~equently u6ea to detect ~rabino6e ~n the ANA p obe.
P. Detection of ~ybr~d~ `
The hybri~ized ~ample would be incubat~a i~ a blocking buf~er whic~ con~ains reagen~6 to reduce ~on-~pecific ad60rption of the antibody-enzy~e conjugate to the membrane. Typically, ~ buff0r ~ontaining lt bovine ~eru~ albu~in i~ u~ed. Followin~ ~eat~Qnt with the blocking bu~fer, the membrane would be i~ubat~d with a~ appropria~0 antibody-~n2y~e ~o~uga~ ~or a 2~ period of ti~e su~h that the ~ntlbo~y ha6 ~n oppor-~unity to recognize a~d bind ~o ~ach arabi~o6e ~oi~ty in the stranD~ of probe ~ybrld~z~d to earget ~u~121c ~cid. ~he antibody-~nzy~e co~3u~a~e ~oul~ eon~t o~
an enzy~e coval~ntly linke~ ~o ~n ~n~lboay or ra~men~
thereo~ which ~ ~p~ciflc ~or arabinose in th~ probe.
Following bindin~ of ehe anei~oay-enzy~e con~ugat~ to the prob~. ~be ~embLan~ whi~h ~ow contaln~ con~ugate boun~ to ~h~ probe ~ybridl2e~ to the target nueleic aoid, i~ wa~hed ~gain ~i~h a 6erie~
of buffer6 ~o re~ove unbound and non6peci~ically bound .
:
6~
con~uqate f~om the ~upport. The appropriate enzyme 6ub6trate an~/or ~hromogen ~6 ln~uba~ed ~ith ~he product on the 6upport ~nd color development ~
allowed to proceed ~or a pre6crlbed perio~ of l;$me.
5 The extent oE hybridization could be ~u~nti~ea by mea6uring the rat~ of color ~evelopment or the total color developed a~ter a Bet time period.
Claims (3)
1. A probe for the detection of a preselected nucleic acid sequence comprising single stranded arabinonucleic acid consisting essentially of arabinonucleotides having 3' and 5' internucleotide linkages with a base linked at the 1' position, said base being selected from the group consisting essentially of adenine, guanine, cytosine, thymine, and uracil, said bases having a sequence complementary to said preselected nucleic acid sequence.
2. A method for identifying nucleic acid sequences comprising the steps of:
(a) rendering the target nucleic acids single-stranded;
(b) immobilizing the single-stranded nucleic acids onto a support;
(c) allowing said single-stranded nucleic acids to hybridize with a single-stranded arabinonucleic acid probe, said arabinonucleic acid consisting essentially of arabinonucleotides having 3' and 5' internucleotide linkages with a base linked at the 1' position, said base being selected from the group consisting essentially of adenine, guanine, cytosine, thymine, and uracil;
(d) washing said support to remove arabinonucleic acid not incorporated into the hybrid formed on the support; and (e) determining the presence of arabinonucleic acid in the hybrid formed on the support by contacting it with an anti-arabinose antibody-label conjugate and detecting said label.
(a) rendering the target nucleic acids single-stranded;
(b) immobilizing the single-stranded nucleic acids onto a support;
(c) allowing said single-stranded nucleic acids to hybridize with a single-stranded arabinonucleic acid probe, said arabinonucleic acid consisting essentially of arabinonucleotides having 3' and 5' internucleotide linkages with a base linked at the 1' position, said base being selected from the group consisting essentially of adenine, guanine, cytosine, thymine, and uracil;
(d) washing said support to remove arabinonucleic acid not incorporated into the hybrid formed on the support; and (e) determining the presence of arabinonucleic acid in the hybrid formed on the support by contacting it with an anti-arabinose antibody-label conjugate and detecting said label.
3. The method of Claim 2 wherein the label is an enzyme.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US812,574 | 1985-12-23 | ||
| US06/812,574 US4760017A (en) | 1985-12-23 | 1985-12-23 | Arabinonucleic acid probes for DNA/RNA assays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1296663C true CA1296663C (en) | 1992-03-03 |
Family
ID=25210011
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000525772A Expired - Lifetime CA1296663C (en) | 1985-12-23 | 1986-12-18 | Arabinonucleic acid probes for dna/rna assays |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US4760017A (en) |
| EP (1) | EP0227459B1 (en) |
| JP (1) | JPS62158299A (en) |
| CA (1) | CA1296663C (en) |
| DE (1) | DE3688931T2 (en) |
Families Citing this family (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4760017A (en) * | 1985-12-23 | 1988-07-26 | E. I. Du Pont De Nemours And Company | Arabinonucleic acid probes for DNA/RNA assays |
| GB8800702D0 (en) * | 1988-01-13 | 1988-02-10 | Nycomed As | Test method & reagent kit therefor |
| US5082830A (en) * | 1988-02-26 | 1992-01-21 | Enzo Biochem, Inc. | End labeled nucleotide probe |
| US5824796A (en) * | 1988-09-28 | 1998-10-20 | Epoch Pharmaceuticals, Inc. | Cross-linking oligonucleotides |
| USRE38416E1 (en) | 1988-09-28 | 2004-02-03 | Epoch Biosciences, Inc. | Cross-linking oligonucleotides |
| US5849482A (en) * | 1988-09-28 | 1998-12-15 | Epoch Pharmaceuticals, Inc. | Crosslinking oligonucleotides |
| US6005087A (en) * | 1995-06-06 | 1999-12-21 | Isis Pharmaceuticals, Inc. | 2'-modified oligonucleotides |
| US5859221A (en) * | 1990-01-11 | 1999-01-12 | Isis Pharmaceuticals, Inc. | 2'-modified oligonucleotides |
| US5459255A (en) * | 1990-01-11 | 1995-10-17 | Isis Pharmaceuticals, Inc. | N-2 substituted purines |
| US6395492B1 (en) * | 1990-01-11 | 2002-05-28 | Isis Pharmaceuticals, Inc. | Derivatized oligonucleotides having improved uptake and other properties |
| US5852182A (en) * | 1990-01-11 | 1998-12-22 | Isis Pharmaceuticals Inc. | Thiol-derivatized oligonucleosides |
| DE69133405T2 (en) * | 1990-01-11 | 2005-07-07 | Isis Pharmaceutical, Inc., Carlsbad | Oligonucleotide derivatives for detecting and modulating RNA activity and gene expression |
| US5872232A (en) * | 1990-01-11 | 1999-02-16 | Isis Pharmaceuticals Inc. | 2'-O-modified oligonucleotides |
| US6399754B1 (en) * | 1991-12-24 | 2002-06-04 | Isis Pharmaceuticals, Inc. | Sugar modified oligonucleotides |
| US6114513A (en) * | 1990-01-11 | 2000-09-05 | Isis Pharmaceuticals, Inc. | Thiol-derivatized oligonucleotides |
| US6339066B1 (en) | 1990-01-11 | 2002-01-15 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotides which have phosphorothioate linkages of high chiral purity and which modulate βI, βII, γ, δ, Ε, ζ and η isoforms of human protein kinase C |
| US6013431A (en) * | 1990-02-16 | 2000-01-11 | Molecular Tool, Inc. | Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators |
| US6262241B1 (en) * | 1990-08-13 | 2001-07-17 | Isis Pharmaceuticals, Inc. | Compound for detecting and modulating RNA activity and gene expression |
| US5177196A (en) * | 1990-08-16 | 1993-01-05 | Microprobe Corporation | Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof |
| US5185264A (en) * | 1990-11-09 | 1993-02-09 | Abbott Laboratories | Diluent buffer and method for diluting an assay component |
| US6004744A (en) | 1991-03-05 | 1999-12-21 | Molecular Tool, Inc. | Method for determining nucleotide identity through extension of immobilized primer |
| US6136601A (en) * | 1991-08-21 | 2000-10-24 | Epoch Pharmaceuticals, Inc. | Targeted mutagenesis in living cells using modified oligonucleotides |
| US6537973B1 (en) | 1992-03-16 | 2003-03-25 | Isis Pharmaceuticals, Inc. | Oligonucleotide inhibition of protein kinase C |
| AU2320597A (en) | 1996-03-19 | 1997-10-10 | Molecular Tool, Inc. | Method for determining the nucleotide sequence of a polynucleotide |
| US7812149B2 (en) | 1996-06-06 | 2010-10-12 | Isis Pharmaceuticals, Inc. | 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations |
| US5898031A (en) | 1996-06-06 | 1999-04-27 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides for cleaving RNA |
| US9096636B2 (en) | 1996-06-06 | 2015-08-04 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
| GB9624165D0 (en) * | 1996-11-19 | 1997-01-08 | Amdex A S | Use of nucleic acids bound to carrier macromolecules |
| US6232463B1 (en) | 1997-10-09 | 2001-05-15 | Isis Pharmaceuticals, Inc. | Substituted purines and oligonucleotide cross-linking |
| AU2001276691A1 (en) * | 2000-08-03 | 2002-02-18 | Matsushita Electric Industrial Co., Ltd. | Brushless motor and method of manufacturing the brushless motor |
| AU2003273336A1 (en) * | 2002-09-18 | 2004-04-08 | Isis Pharmaceuticals, Inc. | Efficient reduction of target rna's by single- and double-stranded oligomeric compounds |
| CA2504929C (en) | 2002-11-05 | 2014-07-22 | Charles Allerson | Compositions comprising alternating 2'-modified nucleosides for use in gene modulation |
| US8569474B2 (en) | 2004-03-09 | 2013-10-29 | Isis Pharmaceuticals, Inc. | Double stranded constructs comprising one or more short strands hybridized to a longer strand |
| CA2569419A1 (en) * | 2004-06-03 | 2005-12-22 | Isis Pharmaceuticals, Inc. | Double strand compositions comprising differentially modified strands for use in gene modulation |
| US8394947B2 (en) | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
| US20060040258A1 (en) * | 2004-08-23 | 2006-02-23 | Huiyan Guo | Water-soluble conjugates and methods of preparation |
| US7884086B2 (en) | 2004-09-08 | 2011-02-08 | Isis Pharmaceuticals, Inc. | Conjugates for use in hepatocyte free uptake assays |
| US20060205090A1 (en) * | 2005-03-14 | 2006-09-14 | Newton Michael W | Water-soluble conjugates for electrochemical detection |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4093714A (en) * | 1974-03-15 | 1978-06-06 | Icn Pharmaceuticals, Inc. | 9β-D-Arabinofuranosylpurine nucleotides and method of use |
| JPS58221168A (en) * | 1982-06-17 | 1983-12-22 | Yamasa Shoyu Co Ltd | Quantitative determination of arabinonucleoside |
| US4556643A (en) * | 1982-07-26 | 1985-12-03 | Agracetus | Assay method and probe for polynucleotide sequences |
| EP0144913A3 (en) * | 1983-12-12 | 1986-08-20 | Miles Inc. | Hybridization assay employing labeled probe and anti-hybrid |
| US4724202A (en) * | 1983-12-12 | 1988-02-09 | Molecular Diagnostics, Inc. | Use of non-hybridizable nucleic acids for the detection of nucleic acid hybridization |
| US4883750A (en) * | 1984-12-13 | 1989-11-28 | Applied Biosystems, Inc. | Detection of specific sequences in nucleic acids |
| JPS62128807A (en) * | 1985-11-30 | 1987-06-11 | Sumitomo Rubber Ind Ltd | Builtup body of run-flat tire for atv and rim |
| US4760017A (en) * | 1985-12-23 | 1988-07-26 | E. I. Du Pont De Nemours And Company | Arabinonucleic acid probes for DNA/RNA assays |
| JP2633832B2 (en) * | 1986-05-16 | 1997-07-23 | 住友ゴム工業 株式会社 | Wheel rim and tire assembly |
-
1985
- 1985-12-23 US US06/812,574 patent/US4760017A/en not_active Expired - Lifetime
-
1986
- 1986-12-18 CA CA000525772A patent/CA1296663C/en not_active Expired - Lifetime
- 1986-12-22 DE DE86310006T patent/DE3688931T2/en not_active Expired - Fee Related
- 1986-12-22 EP EP86310006A patent/EP0227459B1/en not_active Expired - Lifetime
- 1986-12-22 JP JP61304070A patent/JPS62158299A/en active Granted
-
1988
- 1988-04-29 US US07/188,544 patent/US5077196A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62158299A (en) | 1987-07-14 |
| EP0227459A2 (en) | 1987-07-01 |
| DE3688931T2 (en) | 1993-12-23 |
| US5077196A (en) | 1991-12-31 |
| US4760017A (en) | 1988-07-26 |
| JPH0529439B2 (en) | 1993-04-30 |
| EP0227459B1 (en) | 1993-08-25 |
| DE3688931D1 (en) | 1993-09-30 |
| EP0227459A3 (en) | 1989-01-25 |
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