DNA/RNA Aptamer Microarray Service

Mapping a specific binding sequence of an aptamer oligonucleotide to decrease the molecule size for many practical applications such as drug molecules

• Identification of key positions of a consensus sequence for enhancing binding affinity (possibly by modifying those positions) for an aptamer drug candidate.
• Through this strategy, we can map a small but highly specific binding aptamer oligonucleotide.

Background

• Macugen (pegaptanib sodium) is an ophthalmic aptamer drug being developed by Pfizer/Eyetech for the treatment of the 'wet' forms of age-related macular degeneration (AMD).
• The consensus sequence is CGGAAUCAGTGAATGCTTATACATCCGt, an RNA 28 mer where all pyrimidines are modified with a 2'-F and the all the purines (except 2 As) are modified with a 2'-'OMe group.
• Our aptamer chip is a powerful tool for further refining the consensus sequences identified from the in vitro evolution (SELEX), by finding the minimal length of oligonucleotide required for specific binding (core sequence) and systematic modification of the core sequence. The above example shows a strategy for searching the region of core sequence, which will show highest binding signals.

Sequence Design Example

• 3' End Truncation

We can start by truncating the sequence at the 3' end, removing one base at a time.

CGGAAUCAGTGAATGCTTATACATCCG
CGGAAUCAGTGAATGCTTATACATCC
CGGAAUCAGTGAATGCTTATACATC
CGGAAUCAGTGAATGCTTATACAT
CGGAAUCAGTGAATGCTTATACA
CGGAAUCAGTGAATGCTTATAC
CGGAAUCAGTGAATGCTTATA
CGGAAUCAGTGAATGCTTAT
CGGAAUCAGTGAATGCTTA
CGGAAUCAGTGAATGCTT
CGGAAUCAGTGAATGCT
CGGAAUCAGTGAATGC
CGGAAUCAGTGAATG
CGGAAUCAGTGAAT

 

• 5' End Truncation

Next, truncate the sequence at the 5' end, removing one base at a time.

GGAAUCAGTGAATGCTTATACATCCGt
GAAUCAGTGAATGCTTATACATCCGt
AAUCAGTGAATGCTTATACATCCGt
AUCAGTGAATGCTTATACATCCGt
UCAGTGAATGCTTATACATCCGt
CAGTGAATGCTTATACATCCGt
AGTGAATGCTTATACATCCGt
GTGAATGCTTATACATCCGt
TGAATGCTTATACATCCGt
GAATGCTTATACATCCGt
AATGCTTATACATCCGt
ATGCTTATACATCCGt
TGCTTATACATCCGt
GCTTATACATCCGt


• 3' & 5' End Truncation

Next remove two bases at a time, one from either end.

GGAAUCAGTGAATGCTTATACATCCG
GAAUCAGTGAATGCTTATACATCC
AAUCAGTGAATGCTTATACATC
AUCAGTGAATGCTTATACAT
UCAGTGAATGCTTATACA
CAGTGAATGCTTATAC
AGTGAATGCTTATA


 

• 14 mer Frame Walking

Last, we set the sequence length at 14 mer starting from the 5' end and move the frame down the sequence (toward 3' end) as far as we can.

CGGAAUCAGTGAAT
   GGAAUCAGTGAATG
      GAAUCAGTGAATGC
         AAUCAGTGAATGCT
            AUCAGTGAATGCTT
               UCAGTGAATGCTTA
                  CAGTGAATGCTTAT
                     AGTGAATGCTTATA
                        GTGAATGCTTATAC
                           TGAATGCTTATACA
                              GAATGCTTATACAT
                                  AATGCTTATACATC
                                     ATGCTTATACATCC
                                        TGCTTATACATCCG
                                           GCTTATACATCCGT

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