Study on the M ethod of the Qua lita tive Ana lysis of Phosphoroth ioa te O ligonucleotide Can tide

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1 ISSN CN gQ Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy 17 (2): ,, (, ) (A CTCA CTCA GGCCTCA GA CT ),,,,,,,,,,,,, R 917 Study on the M ethod of the Qua lita tive Ana lysis of Phosphoroth ioa te O ligonucleotide Can tide YAN G B ing2hu, GUAN W ei, W AN G Sheng2qi 3 (Institu te of R ad iation M ed icine, A cad emy of M ilitary M ed ical S ciences, B eij ing , Ch ina) Abstract Can tide (A CTCA CTCA GGCCTCA GA CT ) is an an tisen se inh ib ito r again st the act ivity of telom era se. To study the m ethod of the qua lita t ive ana ly sis, an ion exchange h igh perfo rm ance liqu id ch rom atography and cap illary gel electropho resis w ere u sed. T he pho s2 pho ro th ioate o ligonucleo tide and its analogs of deletion sequences as w ell as partial pho sphodiester com pounds w ere analyzed. T he resu lts show ed that an ion exchange H PL C w as sen sitive to pho spho ro th ioate m odification and fu ll pho spho ro th ioate and partial pho sphodiester o ligonucleo tides of the sam e length cou ld be reso lved. T he reten tion tim e is decreased w ith the increasing num ber of pho sphodiester bondṡ A n ion exchange H PL C w as relatively in sen sitive to the length of pho spho ro th ioate o ligonucleo tides, n and n21 sequence cou ld no t be reso lved. CGE w as excellen t fo r separating the deletion sequences from the fu ll2length sequence, bu t it cou ld no t separate pho spho ro th ioate and partial pho sphodiester o ligonucleo tides of the sam e length. Pu rity and m odified degree of pho spho ro th ioate o ligonucleo tides cou ld be determ ined by an ion exchange H PL C and CGE. Key words can tide, an tisen se o ligonucleo tides, telom erase, an ion exchange h igh perfo rm ance liqu id ch rom atography, cap illary gel electropho resis : , : (N o ) (N o ) 915 (N o. 96Z007) 3 T el: (010) , Fax: (010) , E2m ail: sqw ang@nic. bm i. ac. cn, ,, Received: July 6, 2000; A ccep ted: Sep tem ber 21, 2000 Suppo rted by N ational H igh T echno logy 863 P ro ject of Ch ina (N o ), N ational N atural Science Foundation of Ch ina (N o ) and M ilitary 9 5 Key P ro ject (N o. 96Z007) 3 Co rresponding autho r T el: (010) , Fax: (010) , E2m ail: sqw ang@nic. bm i. ac. cn

2 2 : 251 RNA ; CPG, A pp lied B io system s ;,, Beaucage ;, N, [ 1, 2 ],, Sigm a ;,,,,,,,, ; 6 h,, 6 h 20, :,, M illi2q, G5, 114 n- 1 n 391DNA, n- 2, n CPG 80-3 n 90 m in, : a. DM T, 20%, (PA GE) [ 3 ] ; b. (CGE) [ 3 5 ] ; c m l, (H PL C) [ 5 ] ; d. (H PTL C) [ 6 ] ; e. (M S) [ 7, 8 ] ; f. 31 P ( 31 P NM R ) [ 9 ] H PL C, H PL C 20%, CGE, H PL C CGE 115, M illi2q,, 260 nm, 33 gg 1 (n), n - 1 (n - 1), n (n- 2), (O 1), D elta P rep 4000, 2487 (O 2), (O 3), W aters ; 391, T ab le , DNA, A pp lied B io system s ; 01116, 01588, 01578, 01184, ggl PgA CE M DQ, Beckm an 116 ; D u640, Beckm an ;M ill2q B iocel, M illipo re PORO S ( 416 mm 100 mm ), SOU RCE O ligo P rep op 120, 15Q, 15 m A : 10 mm o lgl N aoh, 011 Savan t mm o lgl N ac l; B: 10 mm o lgl N aoh, 2 m o lgl 112 N ac l T ab le 1, 260 nm, PORO S, 416 mm AU FS , 100 mm, Boeh ringer M annheim Gm bh Table 1 T he gradient used in anion exchange H PL C ; SOU RCE15Q, 15 m, Pharm acia B io tech ; M icropu re g, So lid Phase Sciences ; eca P ssdna 1002R DNA Beckm an 113, DNA :, C rauchem T im e gm in F low rate gm l m in - 1 M obile phase compo sition %A %B

3 , 254 nm 100 m, 31 cm, 20 cm, - 10 kv, 2 s kv ph 815 T ris2 27 m o lgl eca P ssdna 1002R DNA, , 3 l, T ab le 2 T ab le 2,,, Table 2 T he retention tim e of Cantide and its analogs N am es Sequence Retention tim egm in n A CTCA CTCA GGCCTCA GA CT n- 1 CTCA CTCA GGCCTCA GA CT n- 2 TCA CTCA GGCCTCA GA CT O 1 A CTCA CTCA GGCCTCA GA CoT O 2 A CTCA CTCA GGCCTCA GA ocot O 3 A CTCA CTCA GGCCTCA GoA ocot N o te: o rep resents P= O link F ig. 1 A nalysis of a m ixture containing mono2, di2, and tripho sphodiester analogs (a) A nion exchange ch rom atogram, (b) Cap illary gel electropherogram,, n n- 2, n n , F ig. 2 (a) F ig. 2 (b) n- 2 n- 1 n 1 2 2, CGE H PL C 212, n n - 2, O 1 O 2 O CGE 1 2 2, H PL C, 3 l, F ig a CGE, n O 1 O 2 O F ig. 1 b. F ig. 1 a H PL C,, H PL C, 3 l, F ig. 3 H PL C F ig. acge, 1 b,, F ig. 3 bf ig. 3 a n O 1 O 2 O 3, H PL C H PL C, CGE,, CGE H PL C CGE n n- 1 n- 2,, 3 l, n n- 1 n- 1 n- 2,,

4 2 : 253,,,, F ig. 4,, F ig. 2 (a) A nion exchange ch rom atogram of a m ixture of full2length and (n- 2) deletion sequence; (b) Cap illary gel electropherogram of full2length and (n- 1), (n- 2) deletion sequences F ig14 T he anion exchange ch rom atogram of o ligonucl2 eo tide pho spho ro tioate p roduction using different so lutions of sulfurizing agent (a) The chrom atogram of the synthetic p roduction used the freshly p repared so lution of sulfurizing agent; ( b ) The chrom atogram of the synthetic p roduction used the so lution of sulfurizing agent stayed fo r three days 3 F ig. 3 A nalysis of full2length, fully th ioated, 202base 5 2 A CTCA CTCA GGCCTCA GA CT 23 and its mono2, di2, and tripho sphodiester analogs (a) A nion exchange chrom atogram; (b) Cap illary gel electropherogram m RNA gvrna, DNA RNA, DNA A SODN, [ 10, ], [ 11, A SODN ],

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