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Dissociation constunts for DNA aptanaersfor detection of II. inJtuenzue type b

Fateme sadat Bitaraf I.Irai Rasooli*r, Seyed Latif Mousavi Gargari"r Department of Biology, Faculty of Sciences, Shahed (htiversity, _Tehran,Iran

E-m ail : [email protected] E-mail":[email protected]

Introduction

Haemopltilus influenxae type b (Hib) causes Acute Bacterial Meningitis

(ABM) in

children

with

the

mortality

rate of about 3- 6oh of the affected patients(l). Aptamers, the short single stranded oligonucleotides

@NA

or RNA)

with

high

affinity

to target

molecule, are selected by a

high-flux

screening technique known as systematic evolution of exponential enrichment technology

(SELEE(2).

The dissociation constant is commonly used to describe

tightly

a aptamer binds to a

particular Hib

cell(3).

Materials

&

Methods

Aptamers 45 and 63 were selected

for further

binding characterization because of

their

high binding

affinity

to H.influenzae Upe ^b during the

preliminary

screening. Each bacterial cell was prepared

with

108 cells using the same SELEX procedure and was incubated

with

different concentrations (0, 10, 50, 100,200, and 300

pM

as

final

concentrations) of fluorescein-labeled ssDNA in 0.5m1 of trinding

buffer for

45 min at

4'C

with mild

shaking. The cells were washed to remove unbound ssDNA

from

cells by centrifugation (8000 g

for

¹⁰min) and were then resuspended

in lml

of binding buffer.

FinallS

the fluorescence intensity of each sample was measured by a

fluorospectrophotometer. To estimate the

affinity

(Kd) of the selected ssDNA to target cells, the fluorescence intensity versus the added ssDNA concentration was plotted.

These data showing saturation curves were

fitted

by nonlinear regression analysis.

Median effective concentration was calculated using the equation

Y:BmaxX/(Kd+X).

_The

equilibrium

dissociation constant

(Kd)

was calculated by

plotting

the average total percentage

of

fluorescent bacterial cells (Y), which corresponded to aptamer-bound

H.inflaenzae

We

^D,^againstthe concentration of aptamer (X) _usinga non-interacting binding sites model

in

SigmaPlot 12.0 (Jandel, San Rafel, CA, USAX4).