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Gutunganya no gukoresha aptameri za aside nucleic

2025-10-08

IntangiriroAPTAMER

Aptamers ni molekile za ADN cyangwa RNA zifite umugozi umwe ziboneka binyuze mu gusuzuma muri vitro, zishobora kwihuza neza n’ibintu bitandukanye (iyoni z’icyuma, poroteyine, uturemangingo). Urufatiro rwazo rwo kumenya molekile ruri mu kwivanga mu miterere yihariye y’ibice bitatu—nk’imirongo y’inyuma, G-quadruplexes na pseudoknots—binyuze mu miterere ya nucleotide. Kwihuza bigerwaho binyuze mu guhuza hydrogen, gukorana kwa electrostatic, imbaraga za van der Waals, n’ingaruka za hydrophobic, hamwe n’ibice bihuza bigera ku rugero rwa nanomolar kugeza kuri picomolar (nM-pM).

Gutunganya no gukoresha aptamers za aside nucleic + KMD Bioscience Fig1

Ishusho ya 1 Ishusho y'ibintu bigira ingaruka ku ntsinzi mu gusuzuma SELEX

Ugereranije n’uturemangingo tw’umubiri, aptamers zitanga ibyiza birimo ubushyuhe buhamye, gukora ibintu mu buryo bugenzurwa, no kutagira itandukaniro ry’ibintu mu buryo bugaragara. Ariko, uburyo bwo guhuza intego, imiterere y’imiterere y’ibintu, no gusaba ubwoko butandukanye bw’ibitabo byose hamwe bisaba isuzuma ryimbitse. Ibi bisaba gushyiraho uburyo bwo gusuzuma bushingiye ku ikoranabuhanga rya SELEX hamwe no kugenzura neza ibintu bihinduka mu bidukikije, bigatanga inkunga y’ibanze mu guteza imbere aptamers zikora cyane.

Ohereza ikibazo

Twandikire Kubindi Bishoboka Wifuza Kumenya Birenzeho Dushobora kuguha igisubizo, Ku bibazo bijyanye n'ibicuruzwa na serivisi byacu. Nyamuneka dusigire imeri yawe kandi tuzagusubiza mu masaha 24.

Kanda kugira ngo ubaze

Ikoranabuhanga ryo gusuzuma SELEXAPTAMER

Inzira nyamukuru y'ikoranabuhanga rya SELEX itangirira ku kubaka isomero rya aside nucleic random rifite umugozi umwe. Isomero rya mbere rya random rifite umugozi umwe rikorwa binyuze mu kongeramo uturere duhoraho tw’ibanze ku mpera zombi no gushyiramo nucleotides 40-60 zidasanzwe hagati. Iri somero rigizwe n'umubare munini w'uruhererekane, rurimo molekile zigera kuri 1014 kugeza 1015 zitandukanye, bikerekana ko uruhererekane rutandukanye cyane. Mu gihe cyo gusuzuma, ibintu nk'imbaraga za ionic zifatana, pH, cations za divalent, n'ubushyuhe birabanza kunozwa. Ibi bikurikirwa n'intambwe yo guhitamo nabi, aho isomero rishyirwamo utudomo twa magnetic, matrices zidafite ikintu, cyangwa imiterere. Ibi bikuraho neza ibimenyetso by'inyuma, byongera umwihariko w'uruhererekane rukurikiraho. Amaherezo, isomero ryakozwe neza rishyirwamo ingero z'icyerekezo zidakora cyangwa zidafite igisubizo zamaze kozwa neza. Uruhererekane rushyirwa hejuru, kandi aptamers za nyuma zishyirwa hanze binyuze mu guhindura ubushyuhe cyangwa reagents za elution.

Ibikoresho bya eleluted bisaba kuvugururwa kwa PCR kw'isomero. Polymerase ya ADN ifite ubushobozi bwo hejuru igomba gukoreshwa mu kongera imbaraga, umubare w'ingendo zo kongera imbaraga ukaguma munsi y'urugero rushoboka. Gel electrophoresis igomba gukorwa kugira ngo igarure imigozi y'ingenzi neza uko bishoboka kose mu gihe hirindwa kwanduzwa na primer dimers, ibicuruzwa byaciwe, cyangwa indi myanda. Ku isomero rya RNA, hongerwaho intambwe yo kwandika muri vitro. Ku isomero rya ssDNA, amasaro ya magnetic apfutswe na streptavidin akunze gukoreshwa mu gutandukanya imigozi. Uru rugendo rwo gufatanya, gukuramo, no kongera imbaraga rusubirwamo inshuro 8-15, rukuraho inyinshi mu ngendo zitari nziza mu gihe rugumana ingero nke z'ingendo nziza. Izo ngendo zemejwe mu isuzuma rya nyuma rya cloning, sequencing, na bindi bigize ingero za nyuma zifuzwa.

Igishushanyo mbonera cy'uruhererekane rwa AptamerAPTAMER

Gupima no kwemeza ibipimo hakoreshejwe NUPACK na CD Spectroscopy

Koresha NUPACK mu gupima ingero za polyconformational (ziruta ingero imwe ya mfold) kugira ngo ubare neza amahirwe yo gukwirakwiza stem-loops, G-quadruplexes, na pseudoknots. Suzuma imiterere ihamye ifunze hamwe n'ingufu zifunguye ≤ -5 kcal/mol, kandi wemeze imiterere y'ingenzi ikoreshwa ukoresheje Circular Dichroism (CD) spectroscopy.

Akarere kagutse gatandukanye kugira ngo hahuzwe ikoreshwa rya iyoni y'icyuma ifite amenyo menshi

Ongera uburebure bw'akarere kadahinduka kugeza kuri 40–60 nt kugira ngo uhuze imiterere y'amabara menshi kuri iyoni z'icyuma, wirinde ko imiterere y'amabara igabanuka bitewe n'imbogamizi za steric mu buryo bugufi (

Gutuza hakoreshejwe Phosphorothioate yihariye n'impinduka za LNA

Shyira neza imiyoboro ya phosphorothioate ku hantu 1-2 ku mpera ya 5' kugira ngo urwanye ukwangirika kwa exonuclease (igipimo cyo guhindura cyagenzuwe na HPLC ≥90%). Gushyira aside Nucleic (LNA) ku byuma by'ingenzi byongera ubushyuhe.

Kwemeza guhuza urubuga hakoreshejwe ITC na SPR

Isothermal titration calorimetry (ITC) ikoreshwa ku gipimo cya aptamer cya ≥5 μM kugira ngo hamenyekane neza impinduka ya enthalpy ihambiriye hamwe n'impinduka ya entropy. Ikoreshwa rya plasmon resonance yo hejuru (SPR) hamwe na biotin-streptavidin immobilization mu gukemura mu buryo bw'igihe nyacyo ibipimo bya kinetic. Hakenewe kwemeza cross-platform hagati ya ITC na SPR, aho ihinduka ry'agaciro gahoraho k'itandukana ry'ikigereranyo rigomba kuba munsi ya 20% kugira ngo hemezwe ko ihambiriye rihagaze neza.

Inyigo ya In Vitro Release Kinetics ku buryo bwo gutanga imiti bukoreshwa mu buryo bwa AptamerAPTAMER

Inyigo za "in vitro release kinetics" za sisitemu zo gutanga imiti ziyobowe na aptamer zigomba gukorwa mu bihe bigenzurwa neza biganisha ku bidukikije by’umubiri. Koresha moderi zikoresha imbaraga cyangwa zidahinduka (agakapu ka dialysis, selile inyuramo, cyangwa uburyo bwo gupima no gutandukanya imiti) kugira ngo upime igipimo n'ingano y'irekurwa ry'imiti mu gice gitwara aptamer. Ibipimo by'ingenzi birimo uburyo bwo kurekura imiti, ubushyuhe, umuvuduko wo guhindagurika, n'igihe cyo gupima. Koresha ultrafiltration centrifugation, chromatography itandukanya ingano, cyangwa gupima ingano y'imiti kugira ngo utandukanye umuti utararekurwa, utandukanye hagati y'umuti wigenga n'umuti uhambiriye. Gerageza gupima ingano y'umuti urekurwa ukoresheje HPLC/UV-Vis cyangwa LC-MS/MS.

Koresha moderi za kinetic cyane cyane ukoresheje ingano ya zero-order, first-order, Higuchi, na Korsmeyer-Peppas. Ku bijyanye no gukwirakwiza kwa Fickian, agaciro ka n ni

Gukoresha Aptamers mu kuvura kanseri itera kanseriAPTAMER

Kugabanuka kwa kanseri ni yo mpamvu nyamukuru itera impfu ziterwa na kanseri, ikaba ikubiyemo intambwe zikurikirana aho uturemangingo twa kanseri dukwirakwira ahantu hashya. Izi ntambwe zitegurwa n'ibikoresho byihariye bigenzura imikorere y'uturemangingo hamwe n'imikoranire y'uturemangingo hagati y'uturemangingo twa kanseri n'ibidukikije bifasha ingingo zitandukanye. Iterambere ry'uburyo bwo kuvura bushingiye kuri aptamer rigaragaza ingamba nziza zo kurwanya kanseri. Uburyo bwo kuvura bushingiye kuri aptamer bukoresha imiti bukorana neza n'inzira z'ibimenyetso mu turemangingo twa kanseri cyangwa itumanaho hagati y'uturemangingo twa kanseri n'ibidukikije bito bya kanseri. Byongeye kandi, aptamers zikora nk'izigamije kugeza imiti ku bidukikije bito bya kanseri. Aptamers zigamije kugaragaza ko zikoreshwa neza mu kuvura kanseri.

Gutunganya no gukoresha aptamers za aside nucleic + KMD Bioscience Fig2

Ishusho ya 2 Ishusho y'icyitegererezo cy'ubuvuzi bushingiye kuri Aptamer bugamije kuvura kanseri hifashishijwe ikoranabuhanga rirwanya kanseri.

Ibibazo Bikunze KubazwaAPTAMER

1. Ni irihe tandukaniro ry'ingenzi riri hagati ya aptameri za aside nucleic na antibodies?

Itandukaniro ry'ingenzi riri hagati ya aptameri za aside nucleic na antibodies riri mu miterere yazo ya molekile, uburyo zikora, n'imikorere yazo. Aptameri za aside nucleic ni molekile za ADN cyangwa RNA zifite umugozi umwe ziboneka binyuze mu buryo bwo gutoranya muri vitro, ubusanzwe zigizwe na nucleotides 20-100. Zifite uburemere buto bwa molekile (6-30 kDa), zishobora gukorwa vuba binyuze mu guhuza imiti, zigaragaza imiterere myiza y’itsinda, kandi zihendutse. Imiterere yazo ifite imiterere itatu ituma zihuza intego runaka, hamwe n'inyungu zirimo ubushobozi bwo kumenya intego zitari poroteyine no kwihanganira ubushyuhe.

Antibodies ni molekile nini za poroteyine (150 kDa) zikorwa n’ubudahangarwa bw’umubiri. Ikorwa ryazo rishingiye ku gukingira inyamaswa cyangwa ikoranabuhanga ry’uturemangingo, bifata amezi kugeza ku myaka, kandi zigira ibyago byo guhinduka mu buryo butandukanye mu gihe bisaba kubika neza mu buryo bukonje. Nubwo antibodies zigaragaza ko zikunda antigen za poroteyine cyane, zirwana no kwibasira ibintu bitari iby’ubudahangarwa bw’umubiri. Byongeye kandi, aptamers zemera impinduka zoroshye za shimi kandi zigaragaza ko zinjira neza mu bidukikije bigoye, mu gihe antibodies zifite ahantu hake ho guhindura zishobora kwangiza imiterere yazo. Nubwo zombi zikora nk'ibikoresho byuzuzanya mu gusuzuma indwara no kuvura, aptamers zongera imbibi z'ibikoresho byo kumenya molekile bitewe n'ubushobozi bwazo bwo kugenzura, guhagarara, no gukoresha neza intego.
2. Ese aptamers zishobora gutandukanya intego zisa mu miterere, nk'ubwoko bwa virusi?

Aptamers zifite ubushobozi bwo gutandukanya intego zisa mu miterere yazo (ubwoko bwa virusi), ariko ubu bushobozi bushingiye cyane ku ngamba zo guhitamo n'itandukaniro rito mu bice by'ingenzi:

(1) Gusobanukirwa neza Epitope no kuyikoresha neza: Gukoresha imbaraga zidasanzwe mu gihe cya SELEX (guhuzanya kw'amarushanwa hamwe n'uburyo butandukanye, guhitamo hagati y'uburyo butandukanye) byongera ubushobozi bwo gukoresha aptamers zigamije epitopes zihariye ku buryo bwihariye;

(2) Guhindagurika kw'Imiterere: Ugereranije n'uturemangingo tw'umubiri, uturemangingo tw'umubiri tworoshye kwimenyera impinduka nto za epitope binyuze mu gusubiza inyuma umugongo (uturemangingo tw'umubiri ...

(3) Kongera ubushobozi bwo kugaragaza ibintu binyuze mu gutunganya tekiniki: Gushyiramo impinduka mu buryo bw’imiterere y’ibintu, imiterere ya nyukleotide yahinduwe hakoreshejwe imiti, cyangwa imiterere ishingiye ku miterere bishobora kunoza ubushobozi bwo kugaragaza ibisigazwa by’ingenzi.
3. Ni izihe ngero z'ikoranabuhanga rya SELEX mu guhitamo aptamer?

Ubwoko butandukanye bwa SELEX bwakozwe kugira ngo bwongere imikorere myiza yo gusuzuma, umwihariko, no gukoresha neza:

(1) Udupira twa Magnetic SELEX: Bibuza ikintu kigomba kwihutishwa (poroteyine cyangwa molekile nto) ku dupira twa rukuruzi, bigatuma habaho gutandukana vuba kw'udupira dufatana hakoreshejwe imbaraga rukuruzi. Ibi bigabanya cyane uruziga rwo gusuzuma kandi bigabanya urusaku rw'inyuma, bigatuma kiba kibereye intego nto za molekile no gusuzuma umusaruro mwinshi.

(2) Seli-SELEX: Ipima aptamers ku buso bw'uturemangingo tuzima, ikarinda imiterere y'umwimerere y'ikintu kigamije guhindura no guhindura nyuma yo guhindura. Ikoreshwa cyane cyane kuri poroteyine zo mu ruhu cyangwa ibimenyetso by'ubuso bugoye (urugero, ku turemangingo twa kanseri). Ariko, bisaba "guhitamo" kugira ngo hakurweho kwihambira kudasanzwe kandi ikubiyemo imiterere ikomeye y'ikoranabuhanga.

Hindura SELEX: Ikoresha uburyo bwo gusuzuma busimburana hamwe n’intego ebyiri zisa. Inzira zo gukurura zihuzwa no gufatanya na Target A idakora zishyirwa ku murongo urwanya Target B kugira ngo zikureho intera zitari izihariye. Gutunganya ibintu mu buryo bw’izunguruka bitanga aptamers zifite umwihariko wo hejuru, zikwiriye gutandukanya poroteyine zihuye cyangwa ubwoko bwa virusi (ubwoko bwa grippe).
4. Ni gute ikoranabuhanga rya SELEX ryarushaho kunozwa mu gusuzuma ikoranabuhanga?

Kugira ngo harebwe imbogamizi za SELEX isanzwe (amasaha maremare yo gusuzuma, gusesengura ibintu byinshi), iterambere riherutse kwibanda ku ngero enye: kubaka urubuga rushingiye kuri chip, kugenzura umuvuduko w'impinduka mu guhitamo, gusesengura amakuru mu buryo bw'ubwenge, no kunoza ubwizigame:

(1) Guhuza ikoranabuhanga rya microfluidic: Amadirishya ya SELEX ashingiye kuri chip ahuza uburyo bwo guhagarika uburyo bwo gukoresha, gutandukanya uburyo bwo gukoresha ikoranabuhanga, no kongera ubushobozi bw'ikoranabuhanga. Igihe cyo gusuzuma inshuro imwe gishobora kugabanywa kugeza ku masaha 2 (kwiyongera inshuro 8 mu buryo bwo gukoresha ikoranabuhanga ugereranije n'uburyo busanzwe) mu gihe bigabanya ikoreshwa ry'ingero.

(2) Igishushanyo mbonera cy'umuvuduko uhindagurika: Inshuro za mbere (1-3) zikoresha icyerekezo cyo hejuru (μM range) kugira ngo zongere ubushobozi bwo gukoresha aptamers. Inshuro zikurikiraho zigabanya buhoro buhoro icyerekezo kugeza ku rugero rwa pM, hamwe n'intambwe zo guhitamo, kugira ngo zongere cyane ubwinshi n'ubunini.

(3) Isesengura ry’Uburyo Bukurikira bwo Gukurikirana (NGS): Gukurikirana uburyo bwo hejuru bukurikirana uko ibintu bigenda mu buryo bwo kongera imiterere y’uruhererekane mu byiciro bitandukanye. Algorithms (AptaCluster) zigaragaza uburyo ibintu bigenda byiyongera cyane, zikirinda gusuzuma ibintu mu buryo budasanzwe.

Impinduka mu bya shimi kugira ngo habeho guhagarara neza: Guhindura phosphorothioate cyangwa 2'-fluoro mu cyiciro cya mbere cya SELEX byongera ubudahangarwa bw'ibitabo mu kwangirika kwa nuclease. Ibi byemeza ko imikorere y'ibice byinshi ikomeza kubaho, cyane cyane ingenzi mu gusuzuma ahantu hagoye nko mu maraso.
5. Ni gute ukuri kw'ibice byatoranijwe bya aptamer kwemezwa?

Kwemeza ukuri bisaba igerageza ry’imiterere myinshi ryemeza ubushobozi bwihariye bwo gufatanya, guhagarara kw’imiterere, n’imikorere:

Koresha Surface Plasmon Resonance (SPR) cyangwa Bio-Layer Interferometry (BLI) kugira ngo umenye neza ingano y'itandukaniro (KD). KD ihuza intego igomba kugera ku rugero rwa nanomolar (ubusanzwe ≤100 nM). Suzuma imikorere y'ihindagurika ry'imiterere y'ihindagurika ry'imiterere y'ihindagurika ry'izuba ukoresheje ELISA cyangwa Fluorescence Polarization, bisaba
Kongeraho intego yigenga byagombye gutuma ikimenyetso cyo gufatanya na aptamer kigabanukaho ≥80%, mu gihe molekile zitagize icyo zivuze zitagomba kugira ingaruka.

Menya ishingiro ry'aho ibintu bihurira ukoresheje uburyo bwo gukata ibice cyangwa guhinduranya aho ibintu biherereye. Komeza imiterere y'ikintu ukoresheje Circular Dichroismspectroscopy cyangwa Small-Angle X-ray Scattering. Emeza ko ibintu bihamye ukoresheje ibizamini bya nuclease resistance.

IndangamuntuAPTAMER

[1] Wu L, Wang Y, Xu X, n'abandi. Gusuzuma intego zikoreshwa mu kuvura neza hakoreshejwe Aptamer. Chem Rev. 2021; 121 (19): 12035-12105.

[2] Kohlberger M, Gadermaier G. SELEX: Ibintu by'ingenzi n'ingamba zo kunoza uburyo bwo guhitamo aptamer neza. Biotechnol Appl Biochem. 2022; 69 (5): 1771-1792.

[3] Alhamhoom Y, As Sobeai HM, Alsanea S, n'abandi. Ubuvuzi bushingiye kuri Aptamer bwo kwibanda ku bintu by'ingenzi bitera kanseri ku mfuruka (Isuzuma). Int J Oncol. 2022; 60(6): 65.