利用高通量CD测量系统和主成分分析来评价g-四联体适配体的结构

DNA适体是与特定分子结合的短DNA分子。由于它们可以通过化学合成产生，并且对靶分子具有高特异性，因此作为药物和生物传感应用的下一代配体，它们一直备受关注。DNA结构的热力学稳定性需要金属离子，金属离子的类型和浓度的变化会导致DNA结构的变化。因此，评估这些金属离子如何影响DNA适体的结构是很重要的。使用JASCO HTCD系统有效地测量了125个样品的CD光谱，并使用PCA方法简单定量地分析了G4适体的结构。该申请说明表明，HTCD系统可以实现对大量样本的自动测量，因此对于G4适体以及其他生物分子的结构特征的全面研究是有效的。Application Note200-CD-037 Structure Evaluation of G-quadruplex Aptamers Using High-ThroughputCD Measurement System and Principal Component Analysis2/4Application Note Structure Evaluation of G-quadruplex Aptamers Using High-Throughpu t CD Measurement System and Principal Component Analysis In t rod u ction DNA aptame r s are short DNA molecules that bind to a speci f ic molecule. Since they can be produced by chemical synthesis and have h igh specifici t y to their target molecules， they have been attracting attention as t he next-gene r ation ligands for pharmaceutical and b iosens i ng appl i ca t i o n s. Metal ions a r e r equired for the thermodynamic stabi li ty of DNA structure， and t h e changes in the type and concen tr ation of metal ions cause changes in DNA structure. It i s therefore i mportant to evaluate how these metal ions effect the structure of DNA aptame r s. Circu l ar dichroism (CD) i s a n effective technique for est i mat in g the tert i ary structure of nucleic acids at l ow concentrations under homogeneous conditions. This appl i cation note reports t h e te r tiary structure evaluation of G-quadruplex (G4)a p tamers using a high-th r oughput CD (HTCD) meas u rement system and t h e principal component a n alys i s (PCA). Keyword s N uc l ei c ac i d me d i c in e ， b i o sen so r， DN A a pt a me r， G -quadru pl ex， G4 a p ta m e r s， circular dich r o i s m ， hi g h -t hr ou g h pu t C D mea s u remen t sy st e m， H T CD， prin c ip al c ompone n t an alysi s ，P C A G4 tertiary structure Four g u a n ine bases ca n associate to form a squ ar e plane r str u ctu r e ca ll ed G-qua r tet， and two o r mo r e G-q u a r tets can stack on eac h othe r t o fo r m a G 4 s tr uc tu re . G 4 i s know n to have th r ee t ypes of tert i ary stru c tu r e：p a r a l le l， hybr i d ， an d an t i-p ara l l e l (Figu r e 1). It i s also k n o w n th at t h e s t r u ctu r e of a G 4 chan g es b e tween t h ese th r ee st r u c t u res a n d the mi x tu r e of th em de p e n di ng o n t h e t yp e o r c onc e nt r a ti o n o f me t a l i o ns . Figure 1. Typ es of G4 tert i a r y str uc t ur e s an d their CD s pe c tr a. CD measurement Measurement system Eight G4 ap tame r s w ith diffe r en t sequences w e r e pr e p a r ed i n f i v e d iff eren t bu f f er s . Eac h sample w as me a s u r ed in tr i p l icate and each buffe r w as meas u red once. In t ota l， 125spe c imens were me a su r ed u sing th e H T CD me a su r e m e n t sy s tem. Features of HTCD measurement system · F u l ly a utom ated f rom sample del i v e ry t o clea n i ng an d dr y in g o f th e f l ow Figure 2. H T CD m ea su r em ent sy s t em.J -1500 is co mb i ne d w i t h HT C D plus . · Up t o 192 specimen s m easu r ed in a si n gle r un · A l l -n ig h t me a su remen ts p o s sibl e Sample Table 1. B u f f er cond it ions In th is appli c ati o n n ote ， th e r e sult s of o ne of th e ei g h t ap ta m e r s m e asur e d ar e shown.T-SO606)(5'-GGGTCGGCTGTCCGTGGGTGGGGA-3') was prepared in 10 m M Tri s -H C I at p H 7.4 wi th d i f fe r e n t s a l t c o nce n tra ti o ns s h o w n i n T a bl e 1.Th e f in a l co n c e n tr a tion of T -SO 606 w a s adjus te d t o 20 p M . Measurement conditions Name NaCl[mM| KCImM| TBS 150 0 TBSK 150 5 TK 0 5 TK150 0 150 Tris 0 0 T h e measuremen t conditions used t o o b ta in th e C D spectra o f T-SO606 are sh o wn i n Ta b le 2. Table 2. Me a s ur e men t co n di t i o nn s Wavelength 320-220nm Data Pitch 0.1nm Bandwidth 1nm Accumulations 4 Response 4 sec Path Length 1nm Scan Speed 50 nm/min Sample Volume 100 pL Analysis T e r tiar y s tr uc tu re o f T-SO606 wa s es ti mated f ro m th e CD spec tr a u sing t h e PCA metho d . 24 r efe r e n ce CD s p ec t ra o f G4s tr uc t ures (Fig u r e 3A ) we r e u s ed t o create a PC A sco r e pl o t sh own in Fi gur e 3B. Th e 23 s p ectra² of the re f eren c e spe c t r a w ere ki n dly p rov i d e d by Dr. Raf a e l d el Villa r -Guerra an d P rof . Jon ath an B . Ch ai res a n d th e ot h e r one was 2KY P 3. Th e score p lo t w as crea t ed using 5 pr in ciple com p onen t s a nd pr o cessed b y nor ma liz in g and cen t e rin g the data . Figure 3. C r e a tion of a PC A sco r e p l o t . (a) R ef eren c e sp e ctr a . Blue， green ， an d r ed co lor ed spec t ra ind i c a t e p a r a ll e l，h y brid ，an d a n t i -paral l e l r e sp e cti v el y. (b) PCA sco re p l o t . Th e e l l ips es i nd i ca t e the 95% con f id e n ce li m i ts of ea c h st r uc t ure. Results Antibody denaturation study The CD spect r a of T -SO606 unde r fi ve di f ferent buffer co n dit i o n s are shown i n Figure 4 and i n dicate th a t t h e spect r al s h ape c h ang e dep e nding on t h e b uf f er cond i t i o n .T -SO606 in buffer co n taining a h i g h NaC l co n c e n t r ation bu t n o KCI (TBS) has p osit i v e p e aks at around 295 nm and 240 nm a n d a n ega ti ve peak at a r ound 260 n m， reminisce n t of G4 ant i-parallel structu r e (F igure 1). T -SO606 i n buffers containing KCI (TBSK， TK， and TK150) h ave a posi ti ve peak at a r ound 260nm and a negative peak at arou n d 240 n m ， sim il ar to G4 p arallel structure (F i gure 1). T -SO606 in a buffer with no NaC I nor KC I (T r i s) has positive peaks at around 285 nm and 255 nm a n d a n ega ti ve peak at a r ound 235 nm. This spectrum i s comparable to the h ybrid structure of G4， however， they have different spectral s h apes. Figure 4. CD sp e ctr a o f T-S O 606 un der f i v e b u ffer co nd it ion s . T he sp e c t r u m w hic h i s d r a w n w i t h a l i ght e r co l o r i n the ba c k gr oun d and t h e co l or ed a re a a round t h e sp e c t r u m i n dica te t he a v er a ge sp e c t r um of e a ch stru c t ur e an d i t s 2o li mi t . B lu e， gre e n and r e d colo r i n d i ca t e p a r a l lel， hybr id and a nti-p a r a l l el res p e c t i v el y . T h e PCA scores of T -SO606 in fi ve differen t buffer cond iti ons are plotted in t h e PCA Score plot i n F i gure 5. Th e PCA scores indicate th at TBS， a buffer co n taini n g high concen t r ation of NaCl only， tends to i nduce an an t i -parallel st r u ct u re of T -SO606， wh e r e as TBSK a n d TK 150， bu ff ers conta i n in g KCl， t e nd to i n duce a paral l el s t r ucture. These considerat i ons agr e e with t h e CD data in Figure 4. Howeve r ， TK， a b uf f er s e emed t o in duce a parallel s t r ucture considering the spectral s h ape， has a score t h at i s ou t of t h e 95 % confidence limit of th e parallel st r uct u re. T he plot i s l ocated between t h e 95%conf i denc e limi t s of the parall el and a n ti-pa r al l e l but st il l close t o t h e paral l el one， w h ich ind i ca t es that TK may ind u ce the equili b rium in which T -SO606 h as a structure similar to a p a r allel s t ructure bu t partially co n tains an a n ti -paral l el structure.The peak feature of T -SO606 in T ri s wa s simi l a r to the one of a hybr i d struc t ure， however， i ts score is plotted b etween t h e 95 % con f idence limits of t he parallel and anti -pa r a ll el. I t is a s sumed th at T ris t e nds to induce the equil i br i um between a paral le l structu r e and a n ant i-paral l el st r uctur e . JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns Figure 5. PC A s co re d o f T-SO606 i n f iv e dif f ere nt bu f f er s . Conclu s ion CD spec tr a of 125 spec i men were ef f ec t ively measu r ed using t h e JASCO HTCD system， and th e structure of G4 aptamers wa s a n alyz e d simply a n d qu a nt i tative l y using th e PCA method. Th is application note demonst r ates t h at HTCD system can a c h ieve au t oma te d mea s u r e ments of a l a r g e n umber of s p ec i men a n d th e refo r e i s effec t ive for comprehe n siv e i n v e stigat i o n of str u ct u ral featur e of G4 a ptam e rs as we l l as ot h e r bio m ol e c u les. R efere n ce s 1. K. Tsukakoshi， K. Abe， K. Sode， K. Ikebuku r o， Anal Chem.，2012，84，5542-5547. 2. R. del Villar Guerra， J. O. Trent ， J . B. Chaires， Ang r ew. Chem. Int. Ed. Engl.，2018，57，7171-7175. 3. V. Kuryavyi， A. T . Phan ， D. J. Patel ， Nucleic Acids Res.， 2010， 38(19)， 6757-6773. This application was deve l o p ed under t h e guidance of Dr. Kaor i Tsukakoshi a n d Prof. Ka z unori Ikebu k uro of Tokyo Unive r sity of Agriculture and T e ch n ology. JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526 A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns