1. health and fitness
2. disease

New Product:
X-Aptamer Selection Kit
Updated April 2015
Technology Basics

X-Aptamers are synthetic affinity molecules


X-Aptamers combine a variety of chemical moieties




Similar in function to monoclonal antibodies
DNA/RNA, amino acid groups, and small molecules
Chemical diversity is far superior to standard aptamers
Improves target interaction for better binding affinity and specificity
A unique bead-based process is used to select X-Aptamers


Only basic molecular biology techniques and equipment required
Enables the X-Aptamer Selection Kit
2
Affinity Molecule Comparison
Key Attributes
Antibodies
Regular Aptamers
X-Aptamers
Affinity
Excellent
Very Good
Excellent
Specificity
Excellent
Very Good
Excellent
High
Low
Low
In Vivo
In Vitro
In Vitro
Refrigerated, Frozen
Ambient
Ambient
Months
Years
Years
Biological
Chemical
Chemical
Low
High
High
Complicated
Simple and Vast
Simple and Vast
Slow
Slow
Rapid
Size & Complexity
Discovery
Storage
Shelf Life
Production
Batch Reproducibility
Functionalizing
Discovery
3
X-Aptamer Chemical Diversity: Examples
Base
R
Phosphorodithioate
X-Aptamers Can Use a Virtually Unlimited Array of Chemical Functionalities
• Incorporated prior to the selection process
• Combinations of multiple functionalities easily accomplished
• Many are incompatible with SELEX (the solution-based aptamer selection process)
4
Increased Chemical Diversity = Better Performance
Target
VEGF165
Regular
Aptamer
KD, unmodified
X-Aptamer
KD, modified
Binding
Affinity
Enhancement
PS2
15 pM
140x
FS2
2.3 pM
913x
Modification
Made
2.1 nM
α-Thrombin
1.1 nM
PS2
1.2 pM
916x
α-Thrombin
2.4 nM
T-indole
1.4 pM
1700x
IgE
11.3 nM
PS2
10.4 pM
1000x
5
New Product: X-Aptamer Selection Kit



Customer

Perform Selection
(up to 3 targets)
Send Sample to
AM Biotech
Identify X-Aptamer
Sequences
AM Biotech

Purchase Kit
<1 week

Enables customer to easily select X-Aptamers
Selection conditions match application
Target identity can remain confidential
Up to 3 targets simultaneously
No special equipment required
Rapid, straightforward, and effective
This is NOT the cumbersome SELEX process!
~3 weeks

Synthesize
X-Aptamers
Send X-Aptamers to
Customer
Customer Uses
X-Aptamers
6
X-Aptamer Selection Kit: Complete Flow
CUSTOMER
AM BIOTECHNOLOGIES
Unique Reagent
Production
Bead Library
Production
Kit
Next Generation
Sequencing
~4 Weeks
Perform Selection
Protocol with up to 3
Desired Targets
Sample
Data Analysis
Synthesize
X-Aptamers
Kit purchase includes a
license for unlimited use of
X-Aptamers for research.
X-Aptamers
material only
Test & Use
X-Aptamer(s)
Licensing terms are available for using X-Aptamers
in a commercial product or service.
7
Kit Testing Results

Prototype X-Aptamer Selection Kits tested


USA, Canada, India, South Africa
100% successful


Every completed kit produced X-Aptamers to at least one target
Each kit could process up to three targets simultaneously

~70% of all targets attempted were successful

Success rate exceeds the SELEX process


SELEX overall success rate estimated to be 30% to 50%
SELEX process is used to select conventional aptamers
8
What Prototype Kit Users Said
“The ease of use [of the kit] allowed my
undergraduate researchers to be able to skillfully
perform the selection experiments, and we have
since identified one of the putative aptamers
identified as a success – this aptamer binds with
low nanomolar affinity and is being used to
generate an electrochemical biosensor platform
for the detection of botulism.” – Dr. Andrew B.,
Metropolitan State University of Denver
“Working in an aptamer research lab, I’m
regularly approached by companies and
researchers from all around the world for
technical help with their aptamer
selections. It seems to me that there has
been a demand for aptamers-on-demand
for quite some time and it is delightful to
see it is finally becoming a reality!” – Dr.
Gwendolyn S., University of Texas at Austin
“Using the prototype kit we were able to
select nanomolar affinity X-Aptamers to a
target candidate biomarker of Tuberculosis
disease…that we failed to raise DNA
aptamers to via conventional SELEX
procedures and we believe that the chemical
modification of the X-Aptamer library was
key to this success.” – Dr. Jonathan B.,
University of Cape Town
“Compared to aptamers selected
through SELEX protocol, X-Aptamers
generated through this kit
demonstrated much higher affinity to
their target molecules on our
platform.” – Dr. Renuka S., University of
North Carolina at Greensboro
9
Kit Pricing – NOT FOR PUBLIC DISCLOSURE
Kit Processing Steps
X-Aptamer Selection Kit Purchase
Price*
Comments
$500 Enables multiplex selection using up to 3 targets
Quality control markers indicate if kit was successfully processed
Next Gen Sequencing (NGS) and Data Analysis
NGS and data analysis by AM Biotech
-orData analysis only by AM Biotech
Customers may choose to obtain NGS from their own vendor/core laboratory
$2,500 Ion Torrent PGM 316/318 chip is preferred platform (FASTQ file required)
-or- Data analysis ranks the sequences for the likelihood of binding to each target
$1,250 Data analysis may also generally indicate sequence specificity to its target
Putative X-Aptamer Synthesis
First 15 (total to all targets attempted)
Second 15 - Optional
Additional (per sequence) – Optional
Synthesize and desalt sequences that data analysis identifies as likely X-Aptamers
$2,250 Putative X-Aptamers can be unlabelled or labeled with either biotin or amine
$1,875 Other labels available for an additional charge
$ 100 X-Aptamer affinity at this stage is expected to be in the nanomolar range
X-Aptamer Enhancement Service – Optional
(performed by AM Biotech)
Commercial use license
Large-scale X-Aptamer Synthesis
Sequence truncation and PS2 backbone modifications to enhance binding affinity
Request 100X to >1000X affinity enhancement is realistic
Quote
Affinity of resulting PS2 X-Aptamers expected to be in the picomolar range
Varies Customer receives a license for unlimited research use of X-Aptamers developed
by
A commercial use license from AM Biotech is required prior to using any X-Aptamer
Market in a commercial product/service or clinical trial
Request AM Biotech can synthesize X-Aptamers at the multi-gram or larger scale
Quote Third party synthesis can be performed after obtaining a commercial use license
* Prices subject to change.
10
Total Price of Kit-based X-Aptamer Selection
$500 Purchase Kit
$2,500 AM Biotech Next Generation Sequencing & Data Analysis
-$1,250
$2,250
AM Biotech Data Analysis Only (customer arranges NGS)
Synthesis of 15 Putative X-Aptamers
(five per target assuming three targets)
$4,000 to $5,250 Total Customer Outlay
$1,333 to $1,750 TOTAL PRICE PER TARGET*
(assuming 3 targets attempted)
* Compared to competitors’ prices of $10,000 to $25,000 per target for turnkey
selection of a conventional aptamer using SELEX .
11
Contacts
Mark Shumbera
President
(832) 295-1483
[email protected]
Tim McGrath
Director, Business Development
(713) 823-2673
[email protected]
AM Biotechnologies, LLC
12521 Gulf Freeway
Houston, Texas 77034-4509
www.am-biotech.com
12
Additional Information
The following slides provide a brief overview of the
technology underlying the X-Aptamer Selection Kit.
13
Basic Components of X-Aptamer Selection
Selection Process
Two-step Bead & Solution Screening Against Targets
Synthesize
Library with
Unique
Reagents
X-APTAMER LIBRARY
Sequencing
Choose &
Synthesize
X-Aptamers
Progression
14
Library: Key Enabler of X-Aptamers and the Kit

A library of billions of different DNA sequences is
synthesized on microbeads
 Total library mass/volume: ~100 mg/0.5 cc
 Sequence diversity: 108 to 1010

Each microbead has ~0.5 picomoles of a unique DNA
sequence attached
300 µm
15
Library Microbead Example
used to help sequence the DNA strand
5’ Primer
5’ Stem
Combinatorial (20-40N)
3’ Stem
3’ Primer
-NNNNNNNNNNNNNNNNNNNNNNNNN-
N = natural or chemically modified nucleotide;
nature of modification is position-dependent
Notional Combinatorial Region on One Bead*
-UTCGAAAUCTGGGACCGUGTTCGUABlack = standard DNA
Green= phosphorodithioate
Red = 2’-OMe phosphorodithioate
Blue = X-modified dU (one or more)
Examples of Available
dU X-Modifications
Indole (Tryptophan)
Phenol (Tyrosine)
Guanidine (Arginine)
Serine
Boron
Small molecules
Others
* Technical Note: The oligonucleotide that includes the chemical modifications shown above cannot be amplified using PCR;
however, an unmodified DNA sequence can be recovered. If this bead is selected from the library, AM Biotech uses the
unmodified DNA sequence recovered after NGS as a ‘barcode’ to re-synthesize the oligonucleotide attached to this bead with the
appropriate chemical modifications in the correct sequence positions.
16
Combinatorial Library Synthesis
A pool and split process (left) creates many copies
of a unique DNA sequence on each bead.
A DNA base is added to each of four synthesis
columns. All the beads in one column get the same
DNA base added. The beads are then removed
from all of the columns, mixed together, and
randomly redistributed back into the four columns.
Another base is added. This process is repeated
numerous times.
This combinatorial process is performed on a
patented instrument and can create as many
unique DNA sequences in parallel as there are
beads available.
17
Bead-based Selection Process
incubate library +
tagged protein
Combinatorial
bead library
(one bead/one
sequence)
protein binds beads
with high affinity
sequences
first stage
magnetic selection
of library beads
anti-tag magnetic
particles bind
protein
recovered
beads (true
& false positives)
cleave sequences from
beads into solution
second-stage
solution pull-down
with target protein
Selection process steps shaded in blue are
performed by the customer using the kit.
Following slides provide additional detail.
next gen
sequencing of
solution pulldown
analyze sequences to
identify X-Aptamers
X
GTG
A A
C T X
AC G
G
C
A
T
X G C
A T
C G
G C X
T
T
A
T
AG
resynthesize highaffinity X-Aptamers
18
Kit Protocol 1st Stage
START KIT
PROTOCOL
Incubate up to 3
tagged targets with
anti-tag magnetic
particles
Add library beads
for selection
Magnetic
selection
of library beads
GO TO NEXT
SLIDE
Isolate magnetically
recovered
beads
Cleave sequences
from
beads into solution
19
Add Target 1
Add Target 2
Add Target 3
No target control
Split into 5
fractions
Isolate binders by magnetic pull-down
CONTINUATION
FROM PREVIOUS
SLIDE
Add anti-tag magnetic particles
Start pool control
PCR amplify with bar-coded primers
Kit Protocol 2nd Stage
Recombine
for next
generation
sequencing
END OF KIT
PROTOCOL
20
Thank You!
Please visit our website.
www.am-biotech.com
21